Antimicrobial and anticancer peptides &amp; conjugates and compositions, methods, articles &amp; kits relating thereto

ABSTRACT

Peptides and conjugates are described herein, including peptides having antimicrobial and/or anticancer properties, as are compositions, articles, and kits comprising such peptides and conjugates, and methods for using the peptides and conjugates.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/710,921 filed Dec. 11, 2019, which claims priority to U.S.Provisional Application No. 62/778,964 filed Dec. 13, 2018, the contentsof each of which are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.HDTRA1-12-C-0039 awarded by the Defense Threat Reduction Agency. Thegovernment has certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (“381789_7000US2_XML”;Size: 63,628 bytes; and Date of Creation: Aug. 13, 2022) is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to peptides and conjugates includingpeptides and conjugates comprising antimicrobial and/or anticancerproperties, to compositions, kits, and articles of manufacturecomprising such peptides and conjugates, as well as to methods for usingthe peptides and conjugates.

BACKGROUND

While a diverse range of therapeutic strategies have been exploredincluding for the delivery of antimicrobial and/or anticancer agents,such strategies often show toxic effect on normal cells. For example,constructs based on chlorine conjugated to polylysines of varied lengthsand varied degrees of substitution have been investigated againstrepresentative gram-negative and gram-positive bacteria for theintracellular delivery of the photosensitizer (chlorine). In thesestudies, cells were treated with the conjugate and then exposed to 660nm light, which triggers the generation of singlet oxygen and freeradicals leading to cell death. Polylysine conjugates are generally notwell suited for systemic administration. They tend to provide limitedspecificity in their delivery of attached drug moieties by entering hostcells, as well as invading microbes. Similar polylysine peptides havebeen used for the transduction of proteins across the membranes ofmammalian cells.

There is a need for new and effective antimicrobial and/or anticanceragents as well as therapeutic, prophylactic, and/or diagnostic methodsand strategies that target microbial organisms and/or cancerous cells.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a peptide comprising:

(a) the amino acid sequence set forth in Formula (I) (SEQ ID NO:1):

X_(aa1) X_(aa2) X_(aa3) X_(aa4) X_(aa5) X_(aa6) X_(aa7) X_(aa8) X_(aa9) X_(aa10) X_(aa11)

wherein independently of each other:

-   -   X_(aa1) is Lys or Arg,    -   X_(aa2) is Lys or Arg,    -   X_(aa3) is Phe, Ala, or Trp,    -   X_(aa4) is Lys or Arg,    -   X_(aa5) is Lys or Arg,    -   X_(aa6) is Phe or Trp,    -   X_(aa7) is Phe or Trp,    -   X_(aa8) iS Lys or Arg,    -   X_(aa9) is Lys or Arg,    -   X_(aa10) is Leu, Phe, or Trp, and    -   X_(aa11) is Lys or Arg; or

(b) the amino acid sequence set forth in Formula (I) (SEQ ID NO:1) withone substitution, insertion, addition, or deletion.

In another aspect, the present invention provides a polynucleotideencoding the peptide of Formula (I) (SEQ ID NO:1).

In other aspects, the present invention provides a compositioncomprising the peptide of Formula (I) (SEQ ID NO:1) or thepolynucleotide encoding the peptide of Formula (I) (SEQ ID NO:1).

In some aspects, the present invention provides an article ofmanufacture comprising the peptide of Formula (I) (SEQ ID NO:1).

In one aspect, the present invention provides a kit comprising thepeptide of Formula (I) (SEQ ID NO:1) or the polynucleotide encoding thepeptide of Formula (I) (SEQ ID NO:1).

In another aspect, the present invention provides a method for treatinginfection by a microbial organism in a subject. The method comprisesadministering to the subject the peptide of Formula (I) (SEQ ID NO:1) orthe polynucleotide encoding the peptide of Formula (I) (SEQ ID NO:1).

In other aspects, the present invention provides a method forpreventing, reducing or inhibiting growth of a microbial organism orbiofilm on a surface. The method comprises contacting the surface with acomposition comprising the peptide of Formula (I) (SEQ ID NO:1).

In some aspects, the present invention provides a method for promotingwound healing in a subject. The method comprises administering to thesubject the peptide of Formula (I) (SEQ ID NO:1) or the polynucleotideencoding the peptide of Formula (I) (SEQ ID NO:1).

In one aspect, the present invention provides a method for treating orpreventing endotoxemia in a subject. The method comprises administeringto the subject an amount of the peptide of Formula (I) (SEQ ID NO:1)effective to treat or prevent endotoxemia in the subject.

In another aspect, the present invention provides a method fordetermining an lipopolysaccharide (LPS) or an lipoteichoic acid (LTA) ina sample. The method comprises contacting the sample with the peptide ofFormula (I) (SEQ ID NO:1) under a condition such that the LPS or the LTAbinds to the peptide to form a complex; and detecting the complex.

In some aspects, the present invention provides a method for diagnosingan LPS- or an LTA-associated disorder in a subject. The method comprisesforming a complex between an LPS or an LTA and the peptide of Formula(I) (SEQ ID NO:1) under a condition such that the LPS or the LTA bindsto the peptide to form the complex; and detecting the complex.

In other aspects, the present invention provides a method for treating acomposition comprising an LPS or an LTA. The method comprises contactingthe composition with the peptide of Formula (I) (SEQ ID NO:1) under acondition such that the LPS or the LTA binds to the peptide to form acomplex; and separating the complex from the composition, therebyreducing or eliminating the LPS or the LTA from the composition.

In some aspects, the present provides a method for determining thepresence of bacteria in a sample. The method comprises contacting thesample with the peptide of Formula (I) (SEQ ID NO:1) under a conditionsuch that the bacteria bind to the peptide to form a complex; anddetecting the complex. The peptide may be in free form or conjugated toan agent or bound to another solid phase.

In other aspects, the present provides a method for isolating bacteriafrom a sample. The method comprises contacting the sample with thepeptide of Formula (I) (SEQ ID NO:1) under a condition such that thebacteria bind to the peptide to form a complex; and isolating thecomplex. The peptide may be in free form or conjugated to an agent orbound to another solid phase.

In one aspect, the present invention provides a method for treating orpreventing a cancer in a subject in need thereof. The method comprisesadministering to the subject a therapeutically or prophylacticallyeffective amount of the peptide of Formula (I) (SEQ ID NO:1), or acomposition comprising the peptide or a polynucleotide encoding thepeptide.

In one aspect, the present invention provides a conjugate comprising thepeptide of Formula (I) (SEQ ID NO:1) conjugated to an agent, wherein thepeptide is connected to the agent directly or through a linker segment,the agent being connected to the peptide or the linker segment through astable or cleavable bond, wherein the conjugate carries and facilitatesthe delivery of the conjugated agent to a microbe or a cancer cell.

In one aspect, the present invention provides a method for targetingdelivery of an agent to a cell in a subject, the method comprisingadministering to the subject the conjugate provided herein.

In some aspects, the present provides a method for determining thepresence of tumor cells in a sample. The method comprises contacting thesample with the peptide of Formula (I) (SEQ ID NO:1) under a conditionsuch that the tumor cell binds to the peptide to form a complex; anddetecting the complex. The peptide may be in free form or conjugated toan agent or bound to another solid phase.

In other aspects, the present provides a method for isolating tumor cellfrom a sample. The method comprises contacting the sample with thepeptide of Formula (I) (SEQ ID NO:1) under a condition such that thetumor cell binds to the peptide to form a complex; and isolating thecomplex. The peptide may be in free form or conjugated to an agent orbound to another solid phase.

In one aspect, the present invention provides a method for increasingthe transferability across cell-membrane of an agent to be delivered toa cell present in a subject the method comprising administering to thesubject the conjugate provided herein.

In other aspects, the present invention provides a method for increasingthe transferability across cell-membrane of an agent to be delivered toa cell ex vivo, the method comprising contacting the cell with theconjugate provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Wimley-White Plot of Hydrophobic Index and Free Energy ofTransfer. The figure depicts how the likeness of the peptide totranslocate from water to POPC membrane interfaces depends on thespecific sequence. The Total Hydrophobic Moment is on the x-axis andFree Energy of Transfer is on the y-axis.

FIGS. 2A-2B are graphs showing endotoxin binding. The figures depict thecompetitive displacement of BC from LPS/LTA by the peptides. 5 μM ofeach peptide was incubated with 10 BC and 10 μg/mL LPS from E. coli(0111:B4) or LTA from S. aureus. Fluorescence intensity was measured atexcitation=580 nm and emission=620 nm. 10 μM BC without endotoxin wasused as 100% displacement while 10 μM BC with 10 μg/mL LPS/LTA withoutany peptide as 0% displacement and the % displacement of BC by eachpeptide was calculated accordingly. PMN had been used as a positivecontrol. The horizontal dotted line coincides with the % displacement ofBC by ATRA1 on the y-axis.

FIG. 3 is a graph showing the antimicrobial efficacy of the peptidesagainst E. coli ATCC 25922 in three different phosphate buffers. Thebuffers were maintained at pH=7.4 and supplemented with 0.1% glucoseprior to use. The buffers were: 10 mM sodium phosphate (blue), PBS(orange) and DPBS (green) and represented as result of triplicate. Whena peptide did not display efficacy in a certain buffer condition, theresults are not shown.

FIG. 4 is a graph showing the antimicrobial efficacy of the peptidesagainst B. cereus ATCC 11778 in DPBS. The buffer (pH=7.4) wassupplemented with 0.1% glucose prior to use.

FIGS. 5A-5B are graphs showing the outer membrane (FIG. 5A) and innermembrane (FIG. 5B) disruptions of E. coli ML35p. Two different phosphatebuffers were used: 10 mM sodium phosphate (blue) and DPBS (green) Theactivity due to HCMAB incubation was considered as 100% disruption whileincubation with no-peptide was considered as 0% disruption.

FIGS. 6A-6B are graphs showing endotoxin (LPS (FIG. 6A) and LTA (FIG.6B)) binding. Competitive displacement of BC from LPS/LTA. In each case,5 μM peptide was incubated with 10 μM BC and 10 μg/mL LPS from E. coli(0111:B4) or LTA from S. aureus. Fluorescence intensity was measuredusing excitation=580 nm and emission=620 nm. As a positive control 10 μMBC without endotoxin was used as a reference for 100% displacement while10 μM BC with 10 μg/mL LPS/LTA without any peptide as a negative controland a reference for 0% displacement. These controls were used tocalculate % BC displacement for each peptide.

FIG. 7 is graph showing anti-microbial effectiveness against type strainE. coli (ATCC 25922) under different conditions of buffer. Various kindsof phosphate buffers were utilized: 10 mM Sodium phosphate (blue), PBS(orange), DPBS (green) and DPBS+4% bovine serum albumin (pink). Thepeptides were also tested against the E. coli in bovine serum (red). Theresults are represented as mean of triplicate. When a peptide did notdisplay efficacy in a certain buffer condition, the results are notshown. LL37 was not tested in 10 mM sodium phosphate buffer or in PBS,and so the results are not shown for those buffers.

FIG. 8 is a graph showing antimicrobial efficacies of ATRA1-R3W2-HYD(blue), NA-CATH (dark orange) and PMB (green) against selected membersof ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiellapneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, andEnterobacter) pathogens. Both susceptible and drug-resistant strainswere used. All the bacteria, except Pseudomonas aeruginosa, were testedunder mammalian serum. P. aeruginosa results were obtained from testingin DPBS. The results are presented as mean of tests in triplicate witherror-bars depicting the 95% confidence interval of the mean.

FIG. 9 is a graph showing the E. coli outer membrane disruption inphosphate buffers with varying ionic composition. Low ionic strength 10mM sodium phosphate (blue) and (physiologically relevant ionicconditions) DPBS (green). The activity due to HCMAB incubation wasconsidered as 100% disruption while incubation with no-peptide wasconsidered as 0% disruption and the results are displayed as mean ofduplicate studies.

FIGS. 10A-10B are graphs showing the hemolysis activity of NA-CATHderived peptide variants and PMB against sheep erythrocytes. Peptideswere incubated at 125 μM against 10% suspension of sheep blood cells inDPBS for 1 hour at 37° C. where, DPBS alone served as negative controland 1% TritonX-100 served as 100% lysis. After incubation, the cells andthe debris were pelleted by centrifugation (1000 r.p.m, 5 min) and thesupernatant was transferred to a microplate and absorbance read at 540nm was conducted. Each peptide was tested in triplicate.

FIGS. 11A-11B are graphs showing cytotoxicity against primary lungepithelium cells in serum-free condition. Cells were seeded in completegrowth medium (Minimal essential medium supplemented with 10% fetalbovine serum) at near-confluence into a 96-well plate and allowed tosettle for 24-hours. Media was then replaced to OptiMEM I reduced serummedium containing peptide or vehicle alone (DBPS) and cells wereincubated overnight (16 hours). A resazurin based dye (Alamar blue) wasadded directly to media and conversion of resazurin to resorufin wasevaluated following two hours. % Survival represents the proportion ofviable cells in peptide treated cells relative to cells treated withvehicle alone. Linear regression was performed to identify theconcentration to achieve 50% lethality (LD₅₀) for each toxic peptide.Each peptide was tested in triplicate.

FIG. 12 is a graph showing comparison of toxicities against Lung PrimaryEpithelial cells and Lung Cancerous Epithelial Cells, derived from H358cell-line. Non-linear regression analysis of six-hour cytotoxicity datawas performed to identify the concentration to achieve 50% lethality(LD₅₀) for each toxic peptide. Each peptide was tested in triplicate.

FIG. 13 is a graph showing perturbation of E. Coli out membrane byNPN-Assay.

FIG. 14 is a graph showing perturbation of E. Coli ML35p out membrane.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are peptides, as well as compositions, conjugates,methods, articles, and kits related to peptides, including antimicrobialpeptides (AMPs) and anti-cancer peptides/constructs, and strategies forleveraging the therapeutic and/or prophylactic potential thereof.According to various aspects and embodiments, the peptides, conjugates,compositions, methods, articles, and kits provided herein can be used,among other things, for targeting a cell (e.g., a microbe, a cancer ortumor cell, a cell containing membranes differing from a normal healthycell, cells with distinct membranes including, for example, cysts andcystic fibrosis), including for targeting a cell for therapeutic and/orprophylactic treatment and/or prevention of cancers and/or ofinfections, wounds and/or biofilms, including infections, wounds and/orbiofilms that involve a microbial organism including, but not limitedto, a microbial organism that may be classified or otherwisecharacterized as a biodefense and/or drug- ormultidrug-resistant/tolerant pathogen.

In some embodiments, the microbial organism is a bacterial strain,virus, fungus, or protozoa.

In one embodiment, the bacterial strain is a Gram-negative orGram-positive bacterial strain.

In another embodiment, the bacterial strain is of the genus Francisela,Acinetobacter, Pseudomonas, Klebsiella, Escherichia, Haemophilus,Proteus, Enterobacter, Serratia, Burkholderia, Stenotrophomonas,Alcaligenes, Mycobacterium, Legionella, Neisseria, Yersinia, Shigella,Vibrio, or Salmonella.

In other embodiments, the bacterial strain is Francisela tularensis,Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae,Klebsiella oxytoca, Escherichia coli, Haemophilus influenzae, Proteusmirabilis, Enterobacter species, Serratia marcescens, Burkholderiacepacia, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans,Mycobacterium tuberculosis, Neisseria gonorrhoeae, Yersinia pestis,Shigella dysenteriae, Vibrio cholera, or Salmonella typhi.

In one embodiment, the bacterial strain is Francisela tularensis,Francisela novicida, Francisela hispaniensis, Francisela noatunensis,Francisela philomiragia, Francisela halioticida, Franciselaendociliophora, Francisela guangzhouensis, or Francisela piscicida.

In another embodiment, the bacterial strain is Francisela tularensis.

In other embodiments, the bacterial strain is of the genusStaphylococcus, Bacillus, Rhodococcus, Actinobacteria, Lactobacillus,Actinomyces, Clostridium, or Streptococcus.

In some embodiments, the bacterial strain is Staphylococcus aureus,Bacillus anthracis, Streptococcus mutans or Streptococcus sanguinis.

In other embodiments, viruses include but are not limited to influenzavirus, parainfluenza virus, respiratory syncytial virus, humanmetapneumovirus, corona virus family members, human immunodeficiencyvirus, herpes simplex virus, cytomegalovirus, SARS (Severe AcuteRespiratory Syndrome) virus, and Epstein-Barr virus.

In some embodiments. fungi include but are not limited to Histoplasmacapsulatum, Coccidioides immitis, Blastomyces dermatitidis,Paracoccidioides brasiliensis, Candida sp., Aspergillus sp., Mucor sp.,Cryptococcus neoformans.

In other embodiments, protozoa include but are not limited to Entamoeba,Acanthamoeba, Balamuthia, Leishmania, Trypanosoma, Trichomonas,Lophomonas, Cryptosporidium, Cyclospora, Toxoplasma, Plasmodium,Babesia, Encephalitozoon, Enterocytozoon and Balantidium.

Some non-limiting examples of cancer include carcinoma, melanoma,lymphoma, blastoma, sarcoma, germ cell tumors, and leukemia or lymphoidmalignancies. Non-limiting examples of cancers that fall within thesebroad categories include squamous cell cancer (e.g., epithelial squamouscell cancer), lung cancer including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung and squamous carcinoma ofthe lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including lung cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, cancer of the urinary tract, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain, aswell as head and neck cancer, and associated metastases.

In other embodiments, cancer also encompasses cell proliferativedisorders which are associated with some degree of abnormal cellproliferation and includes, but not limited to, tumors, which includeneoplasms or neoplastic cell growth and proliferation, whether malignantor benign, and all pre-cancerous and cancerous cells and tissues. In oneembodiment, the cancer is a lung cancer. In some embodiments, the lungcancer is an adenocarcinoma, a squamous cell carcinoma, a large cellcarcinoma, a small cell lung cancer, an adenosquamous carcinoma, or asarcomatoid carcinoma.

In another embodiment, a cell can be a cell containing membranesdiffering from normal healthy cells. In some embodiments, a cell can bea cell with a distinct membrane including, but not limited to, cysts,cystic fibrosis cells, tumor and cancer cells. In other embodiments, thecell comprises a cell membrane having a net-negative chargecharacteristic.

Subjects that can be administered or otherwise benefit from thepeptides, compositions, methods, articles, and kits provided hereininclude vertebrates such as, without limitation, mammals. A mammal canbe a human or animal including livestock and companion animals.Companion animals include but are not limited to animals kept as pets.Examples of companion animals include cats, dogs, and horses, as well asbirds, such as parrots and parakeets. Livestock refers to animals rearedor raised in an agricultural setting to make products such as food orfiber, or for its labor. In some embodiments, livestock are suitable forconsumption by mammals, for example humans. Examples of livestockanimals include mammals, such as cattle, goats, horses, pigs, sheep,including lambs, and rabbits, as well as birds, such as chickens, ducksand turkeys.

In some embodiments, the subject is a human. In another embodiment, thesubject is a non-human mammal.

In other embodiments, the subject can be a human who is a medicalpatient (e.g., a diabetes patient, or a patient in a hospital, clinic),a member of the armed services or law enforcement, a fire fighter, or aworker in the gas, oil, or chemical industry. In one embodiment, thesubject is an animal that is a veterinarian subject/patient (e.g.,livestock or companion animal).

Absent an express indication of the N-terminus and/or C-terminus of apeptide set forth herein, the peptide is to be read from the N-terminusto C-terminus. In some embodiments, individual residues are indicated bythe identity of the amino acid using a standard one- and/or three-lettercode known to one of ordinary skill in the art.

In some aspects, the sequence of a peptide of the present invention canbe based on the sequence or portion of the 34-residue Naja atracathelicidin (NA-CATH) peptide, which corresponds to a helicalcathelicidin identified in cDNA from the venom gland of the elapidsnake, Naja atra (Zhao et al., Peptides 29(10):1685-1691 (2008)).NA-CATH has the sequence KRFKKFFKKLKNSVKKRAKKFFKKPKVIGVTFPF (SEQ IDNO:51) and includes two 11 amino acid repeats (underlined) that differfrom one another at the third and tenth positions (WO 2012/145680; deLatour, F. et al., Biochemical and Biophysical Research Communications396:825-830 (2010)).

In some embodiments, the peptides provided herein can be shorter and/orvariant versions of the NA-CATH peptide, including peptides having oneor more substitutions, insertions, and/or deletions relative to theNA-CATH peptide sequence or a portion thereof. In other embodiments, thepeptides have one or more biological activities (e.g., antimicrobialand/or anticancer and/or bacteria-binding and/or tumor cell-binding).

In some embodiments, the peptides provided herein can include one ormore (e.g., one, two, three, four, five or more) substitutions,insertions, deletions, and/or additions (and combinations thereof) ascompared to the NA-CATH sequence set forth in SEQ ID NO:51.

Amino acid substitutions can be conservative or non-conservative aminoacid substitutions. Conservative amino acid substitutions can be, forexample, aspartic-glutamic as acidic amino acids;lysine/arginine/histidine as basic amino acids; leucine/isoleucine,methionine/valine, alanine/valine as hydrophobic amino acids;serine/glycine/alanine/threonine as hydrophilic amino acids.Conservative amino acid substitutions also include groupings based onside chains. For example, amino acids having aliphatic side chains suchas glycine, alanine, valine, leucine, and isoleucine; amino acids havingaliphatic-hydroxyl side chains such as serine and threonine; amino acidshaving amide-containing side chains such as asparagine, glutamine andcitrulline; amino acids having aromatic side chains such asphenylalanine, tyrosine, and tryptophan; amino acids having basic sidechains such as lysine, arginine, and histidine; and amino acids havingsulfur-containing side chains such as cysteine and methionine.Non-conservative amino acid substitutions typically entail exchanging amember of one of the classes described above for a member of anotherclass. After making an amino acid substitution, insertion, deletion,and/or addition, the activity of a peptide containing the amino acidsubstitution, insertion, deletion, or addition can be assessed using theassays described herein.

In some embodiment, a C-terminal amide, or other C-terminal cappingmoiety can be present in peptides described herein. In one embodiment, apeptide described herein is amidated at the C-terminal.

In other aspects, the present invention provides a peptide comprising:

(a) the amino acid sequence set forth in Formula (I) (SEQ ID NO:1):

X_(aa1) X_(aa2) X_(aa3) X_(aa4) X_(aa5) X_(aa6) X_(aa7) X_(aa8) X_(aa9) X_(aa10) X_(aa11)

wherein independently of each other:

-   -   X_(aa1) is Lys or Arg,    -   X_(aa2) is Lys or Arg,    -   X_(aa3) is Phe, Ala, or Trp,    -   X_(aa4) is Lys or Arg,    -   X_(aa5) is Lys or Arg,    -   X_(aa6) is Phe or Trp,    -   X_(aa7) is Phe or Trp,    -   X_(aa8) is Lys or Arg,    -   X_(aa9) is Lys or Arg,    -   X_(aa10) is Leu, Phe, or Trp, and    -   X_(aa11) is Lys or Arg; or

(b) the amino acid sequence set forth in Formula (I) (SEQ ID NO:1) withone or more substitutions, insertions, additions, or deletions.

In one embodiment, the peptide comprises:

(a) the amino acid sequence set forth in Formula (I) (SEQ ID NO:1); or

(b) the amino acid sequence set forth in Formula (I) (SEQ ID NO:1) withone or more substitutions, insertions, additions, or deletions;

with the proviso that the amino acid sequence is not KRFKKFFKKLK (SEQ IDNO:2), KRAKKFFKKPK (SEQ ID NO:3), or KRAKKFFKKLK (SEQ ID NO:4).

In another embodiment, the peptide comprises the amino acid sequenceKKAKKFFKKLK (SEQ ID NO:5) with one, two, three, four, or fivesubstitutions, insertions, additions, or deletions, with the provisothat the amino acid sequence is not the sequence set forth in SEQ IDNO:2, 3, or 4. In one embodiment, the peptide comprises the amino acidsequence set forth in SEQ ID NO:5.

In some embodiments, the peptide comprises the amino acid sequenceKRAKKFFKRLK (SEQ ID NO:6) with one, two, three, four, or fivesubstitutions, insertions, additions, or deletions, with the provisothat the amino acid sequence is not the sequence set forth in SEQ IDNO:2, 3, or 4. In other embodiments, the peptide comprises the aminoacid sequence set forth in SEQ ID NO:6.

In one embodiment, the peptide comprises the amino acid sequenceKRAKRFFKRLK (SEQ ID NO:7) with one, two, three, four, or fivesubstitutions, insertions, additions, or deletions, with the provisothat the amino acid sequence is not the sequence set forth in SEQ IDNO:2, 3, or 4. In another embodiment, the peptide comprises the aminoacid sequence set forth in SEQ ID NO:7.

In some embodiments, the peptide comprises the amino acid sequenceRRAKRFFKRLK (SEQ ID NO:8) with one, two, three, four, or fivesubstitutions, insertions, additions, or deletions, with the provisothat the amino acid sequence is not the sequence set forth in SEQ IDNO:2, 3, or 4. In other embodiments, the peptide comprises the aminoacid sequence set forth in SEQ ID NO:8.

In one embodiment, the peptide comprises the amino acid sequenceRRFKRFFKRLK (SEQ ID NO:9) with one, two, three, four, or fivesubstitutions, insertions, additions, or deletions, with the provisothat the amino acid sequence is not the sequence set forth in SEQ IDNO:2, 3, or 4. In another embodiment, the peptide comprises the aminoacid sequence set forth in SEQ ID NO:9.

In some embodiments, the peptide comprises the amino acid sequenceRRFRRFFRRLR (SEQ ID NO:10) with one, two, three, four, or fivesubstitutions, insertions, additions, or deletions, with the provisothat the amino acid sequence is not the sequence set forth in SEQ IDNO:2, 3, or 4. In other embodiments, the peptide comprises the aminoacid sequence set forth in SEQ ID NO:10.

In one embodiment, the peptide comprises the amino acid sequenceKRFKKFFKKFK (SEQ ID NO:11) with one, two, three, four, or fivesubstitutions, insertions, additions, or deletions, with the provisothat the amino acid sequence is not the sequence set forth in SEQ IDNO:2, 3, or 4. In another embodiment, the peptide comprises the aminoacid sequence set forth in SEQ ID NO:11.

In some embodiments, the peptide comprises the amino acid sequenceKRWKKFFKKWK (SEQ ID NO:12) with one, two, three, four, or fivesubstitutions, insertions, additions, or deletions, with the provisothat the amino acid sequence is not the sequence set forth in SEQ IDNO:2, 3, or 4. In other embodiments, the peptide comprises the aminoacid sequence set forth in SEQ ID NO:12.

In one embodiment, the peptide comprises the amino acid sequenceKRWKKWFKKWK (SEQ ID NO:13) with one, two, three, four, or fivesubstitutions, insertions, additions, or deletions, with the provisothat the amino acid sequence is not the sequence set forth in SEQ IDNO:2, 3, or 4. In another embodiment, the peptide comprises the aminoacid sequence set forth in SEQ ID NO:13.

In some embodiments, the peptide comprises the amino acid sequenceKRWKKWWKKWK (SEQ ID NO:14) with one, two, three, four, or fivesubstitutions, insertions, additions, or deletions, with the provisothat the amino acid sequence is not the sequence set forth in SEQ IDNO:2, 3, or 4. In other embodiments, the peptide comprises the aminoacid sequence set forth in SEQ ID NO:14.

In one embodiment, the peptide comprises the amino acid sequenceRRWKRFFKRWK (SEQ ID NO:15) with one, two, three, four, or fivesubstitutions, insertions, additions, or deletions, with the provisothat the amino acid sequence is not the sequence set forth in SEQ IDNO:2, 3, or 4. In another embodiment, the peptide comprises the aminoacid sequence set forth in SEQ ID NO:15.

In one embodiment, the peptide is amidated at the C-terminal.

In some embodiments, the peptide further comprises a linked segment. Inone embodiment, the linked segment is a C-terminal tail of the peptide.

In one embodiment, the linked segment is a hydrophobic segmentcomprising one or more hydrophobic moieties such as, for example, one ormore hydrophobic amino acids, etc.

In another embodiment, the linked segment comprises a second amino acidsequence comprising one or more hydrophobic amino acids, for example oneor more of glycine (Gly), alanine (Ala), valine (Val), leucine (Leu),isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met),and/or tryptophan (Trp) and combinations thereof.

In other embodiments, the linked segment is capable of adopting a randomcoil structure. For example, in one embodiment, the linked segmentadopts a random coil structure in the presence of an anionic lipid orliposome.

For example, the last 8-residues of NA-CATH are hydrophobic and adoptrandom coil structures in the presence of anionic liposomes. Withoutwishing to be bound by any particular theory, it is believed that thepresence of unstructured C-terminal tails had demonstrated enhancementof anti-microbial and anti-endotoxin activities in some partiallyhelical peptides.

In one embodiment, the linked segment has a linked segment amino acidsequence set forth in Formula (II) (SEQ ID NO:16): X_(aa1) X_(aa2)X_(aa3) X_(aa4) X_(aa5) X_(aa6) X_(aa7) X_(aa8) X_(aa9) X_(aa10)

wherein independently of each other:

-   -   X_(aa1) is absent or Gly;    -   X_(aa2) is absent or Gly;    -   X_(aa3) is absent, a hydrophobic amino acid, Arg, or citrulline,    -   X_(aa4) is a hydrophobic amino acid, Arg, or citrulline,    -   X_(aa5) is a hydrophobic amino acid, Arg, or citrulline,    -   X_(aa6) is a hydrophobic amino acid, Arg, or citrulline,    -   X_(aa7) is a hydrophobic amino acid, Arg, or citrulline,    -   X_(aa8) is a hydrophobic amino acid, Arg, or citrulline,    -   X_(aa9) is a hydrophobic amino acid, Arg, citrulline, or Tyr,        and    -   X_(aa10) is absent, a hydrophobic amino acid, Arg, or        citrulline.

In some embodiments, the linked segment has the linked segment sequenceset forth in (SEQ ID NO:16), wherein each of X_(aa3)-X_(aa10) is ahydrophobic amino acid.

In other embodiments, the linked segment has the linked segment sequenceVIGVTFPF (SEQ ID NO:17).

In another embodiment, the linked segment has the linked segmentsequence VIGVSIPF (SEQ ID NO:18).

In one embodiment, the linked segment has the linked segment sequenceVIGVTIPF (SEQ ID NO:19).

In other embodiments, the linked segment has the linked segment sequenceGGVIGVTFPF (SEQ Ili NO:20).

In another embodiment, the linked segment has the linked segmentsequence GGVIGVSIPF (SEQ NO:21).

In one embodiment, the linked segment has the linked segment sequenceGGVIGVTIPF (SEQ NO:22).

In some embodiments, the peptide comprises the amino acid sequence setforth in any one of SEQ ID NOs:5-15, wherein the peptide furthercomprises the linked segment sequence as set forth in VIGVTFPF (SEQ IDNO:17).

In other embodiments, the peptide comprises the amino acid sequence setforth in any one of SEQ ID NOs:5-15, wherein the peptide furthercomprises the linked segment sequence as set forth in VIGVSIPF (SEQ IDNO:18).

In other embodiments, the peptide comprises the amino acid sequence setforth in any one of SEQ ID NOs:5-15, wherein the peptide furthercomprises the linked segment sequence as set forth in VIGVTIPF (SEQNO:19).

In some embodiments, the peptide comprises the amino acid sequence setforth in any one of SEQ ID NOs:5-15, wherein the peptide furthercomprises the linked segment sequence as set forth in GGVIGVTFPF (SEQ IDN0:20).

In other embodiments, the peptide comprises the amino acid sequence setforth in any one of SEQ ID NOs:5-15, wherein the peptide furthercomprises the linked segment sequence as set forth in GGVIGVSIPF (SEQII) NO:21).

In other embodiments, the peptide comprises the amino acid sequence setforth in any one of SEQ ID NOs:5-15, wherein the peptide furthercomprises the linked segment sequence as set forth in GGVIGVTIPF (SEQ IDNO:22).

In one embodiment, a peptide described herein is amidated at theC-terminal.

In other aspects, the present invention provides peptide conjugatescomprising other peptides conjugated to a linked segment describedherein. In one embodiment, the linked segment is a C-terminal tail ofthe conjugate. In some embodiments, the linked segment comprises alinked segment sequence set forth in Formula (II) (SEQ ID NO:16). Inother embodiments, the linked segment sequence comprises SEQ ID NO:17,18, 19, 20, 21, or 22. In one embodiment, the linked segment sequencecomprises SEQ ID NO:20, 21, or 22.

In one embodiment, the peptide conjugate is amidated at the C-terminal.

In another embodiment, the linked segment is covalently bonded in thepeptide through a linker. In some embodiments, a linker is optional. Inother embodiments, peptides are provided further having a linker asdescribed herein, covalently linking the linked segment with the aminoacid sequence of the rest of the peptide.

In some embodiments, the linker is a bond.

In one embodiment, the linker is a peptide linker.

In various embodiments, the peptide linker is a short, flexible bridgingsegment.

In some embodiments, the peptide linker is less than about 30 aminoacids, preferably less than about 10 amino acids and more preferablyabout 2, 3, 4, or 5 amino acids. In one embodiment, the peptide linkercomprises 2 amino acids.

In one embodiment, the linker includes from 1 to 30 or less amino acidslinked by peptide bonds. The amino acids can be selected from the 20naturally occurring (i.e., physiological) amino acids. Alternatively,non-natural amino acids can be incorporated either by chemicalsynthesis, post-translational chemical modification or by in vivoincorporation by recombinant expression in a host cell. Some of theseamino acids may be glycosylated. In another embodiment, the 1 to 30 orless amino acids are selected from glycine, alanine, proline,asparagine, glutamine, and lysine, and further from aspartate andglutamate.

Peptide linkers include, without limitation, (shown in single-lettercode): GG; GGG; GGGG (SEQ ID NO:23); GGGGGG (SEQ ID NO:24); GPNGG (SEQID NO:25); SGG; GGSGGS (SEQ ID NO:26); SAT; PVP; PSPSP (SEQ ID NO:27);AAA; ASA; ASASA (SEQ ID NO:28); PSPSPSP (SEQ ID NO:29); KKKK (SEQ IDNO:30); RRRR (SEQ ID NO:31); (G₄S)₃ (SEQ ID NO:32); GGGGS (SEQ IDNO:33); GGGGSGGGGS (SEQ ID NO:34); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:35);GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:36); andGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:37).

In other embodiments, charged linkers may be used. Such charges linkersmay contain a significant number of acidic residues (e.g., Asp, Glu, andthe like), or may contain a significant number of basic residues (e.g.,Lys, Arg, and the like), such that the linker has a pI (isoelectricpoint) lower than 7 or greater than 7, respectively. As understood byone of ordinary skill in the art, and all other things being equal, thegreater the relative amount of acidic or basic residues in a givenlinker, the lower or higher, respectively, the pI of that linker willbe. Such linkers may impart advantageous properties to the peptidesdisclosed herein, such as modifying the peptides pI, which can in turnimprove solubility and/or stability characteristics of such peptides ata particular pH, such as at physiological pH (e.g., between pH 7.2 andpH 7.6, inclusive), or in a pharmaceutical composition including suchpeptides. As is known to one of ordinary skill in the art, solubilityfor a peptide may be improved by formulation in a composition having apH that is at least or more than plus or minus one pH unit from the pIof the peptide.

Amino acid-based linkers can be L form, D form, combinations of L and Dforms, β-form, PEG backbone, and the like.

In one embodiment, the linker comprises a diglycine (i.e., GG).

In another embodiment, the linker is a nonpeptide linker.

Nonpeptide linkers include, without limitation, PEG of any chain length(e.g., —(CH₂—CH₂—O)_(n)— wherein n is such that the PEG linker can havea molecular weight (MW) of about 50 to about 5000 kD, or about 50 toabout 500 kD, or about 50 to about 100 kD); 2-nitrobenzene orO-nitrobenzyl; nitropyridyl disulfide; dioleoylphosphatidylethanolamine(DOPE); S-acetylmercaptosuccinic acid;1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA);β-glucuronide and β-glucuronide variants; poly(alkylacrylic acid);benzene-based linkers (for example,2,5-Bis(hexyloxy)-1,4-bis[2,5-bis(hexyloxy)-4-formyl-phenylenevinylene]benzene)and like molecules; disulfide linkages; poly(amidoamine); carbonnanotubes; hydrazone and hydrazone variant linkers; succinate, formate,acetate butyrate, other like organic acids; aldols, alcohols, or enols;peroxides; alkane or alkene groups of any chain length; one or moreporphyrin or dye molecules containing free amide and carboxylic acidgroups; one or more DNA or RNA nucleotides, including polyamine andpolycarboxyl-containing variants; inulin, sucrose, glucose, or othersingle, di or polysaccharides; linoleic acid or other polyunsaturatedfatty acids; and variants of any of the aforementioned containinghalogen or thiol groups.

In some embodiments, the peptide further comprising the linker and thelinked segment can be chemically synthesized or recombinantly expressedas a fusion protein (i.e., a chimeric fusion protein). One of ordinaryskill in the art knows how to chemically synthesize and/or recombinantlyexpress a fusion peptide or protein.

In other embodiments, the peptide includes the linked segment at theC-terminal, and a linker that is N-terminal to the linked segment,wherein the amino acid sequence set forth in any one of SEQ ID NOs:1-15corresponds to the N-terminal portion of the peptide. In one embodiment,expressly excluded are peptides in which the linked segment is linkeddirectly to any one of SEQ ID NOs:1 and 5-15 without a linker.

In some embodiments, the peptide comprises the sequenceKRFKKFFKKLKGGVIGVTFPF (SEQ ID NO:38), with one, two, three, four, orfive substitutions, insertions, additions, or deletions, with theproviso that the amino acid sequence is not the sequence set forth inSEQ ID NO:2, 3, or 4. In one embodiment, the peptide comprises thesequence KRFKKFFKKLKGGVIGVTFPF (SEQ ID NO:38).

In other embodiments, the peptide comprises the sequenceKRAKKFFKKLKGGVIGVTFPF (SEQ ID NO:39), with one, two, three, four, orfive substitutions, insertions, additions, or deletions, with theproviso that the amino acid sequence is not the sequence set forth inSEQ ID NO:2, 3, or 4. In other embodiments, the peptide comprises thesequence KRAKKFFKKLKGGVIGVTFPF (SEQ ID NO:39).

In one embodiment, the peptide comprises the sequenceKKAKKFFKKLKGGVIGVTFPF (SEQ ID NO:40), with one, two, three, four, orfive substitutions, insertions, additions, or deletions, with theproviso that the amino acid sequence is not the sequence set forth inSEQ ID NO:2, 3, or 4. In one embodiment, the peptide comprises thesequence KKAKKFFKKLKGGVIGVTFPF (SEQ ID NO:40).

In another embodiment, the peptide comprises the sequenceKRAKKFFKRLKGGVIGVTFPF (SEQ ID NO:41), with one, two, three, four, orfive substitutions, insertions, additions, or deletions, with theproviso that the amino acid sequence is not the sequence set forth inSEQ ID NO:2, 3, or 4. In another embodiment, the peptide comprises thesequence KRAKKFFKRLKGGVIGVTFPF (SEQ ID NO:41).

In some embodiments, the peptide comprises the sequenceKRAKRFFKRLKGGVIGVTFPF (SEQ ID NO:42), with one, two, three, four, orfive substitutions, insertions, additions, or deletions, with theproviso that the amino acid sequence is not the sequence set forth inSEQ ID NO:2, 3, or 4. In some embodiments, the peptide comprises thesequence KRAKRFFKRLKGGVIGVTFPF (SEQ ID NO:42).

In other embodiments, the peptide comprises the sequenceRRAKRFFKRLKGGVIGVTFPF (SEQ ID NO:43), with one, two, three, four, orfive substitutions, insertions, additions, or deletions, with theproviso that the amino acid sequence is not the sequence set forth inSEQ ID NO:2, 3, or 4. In other embodiments, the peptide comprises thesequence RRAKRFFKRLKGGVIGVTFPF (SEQ ID NO:43).

In one embodiment, the peptide comprises the sequenceRRFKRFFKRLKGGVIGVTFPF (SEQ ID NO:44), with one, two, three, four, orfive substitutions, insertions, additions, or deletions, with theproviso that the amino acid sequence is not the sequence set forth inSEQ ID NO:2, 3, or 4. In one embodiment, the peptide comprises thesequence RRFKRFFKRLKGGVIGVTFPF (SEQ ID NO:44).

In another embodiment, the peptide comprises the sequenceRRFRRFFRRLRGGVIGVTFPF (SEQ ID NO:45), with one, two, three, four, orfive substitutions, insertions, additions, or deletions, with theproviso that the amino acid sequence is not the sequence set forth inSEQ ID NO:2, 3, or 4. In another embodiment, the peptide comprises thesequence RRFRRFFRRLRGGVIGVTFPF (SEQ ID NO:45).

In some embodiments, the peptide comprises the sequenceKRFKKFFKKFKGGVIGVTFPF (SEQ ID NO:46), with one, two, three, four, orfive substitutions, insertions, additions, or deletions, with theproviso that the amino acid sequence is not the sequence set forth inSEQ ID NO:2, 3, or 4. In some embodiments, the peptide comprises thesequence KRFKKFFKKFKGGVIGVTFPF (SEQ ID NO:46).

In other embodiments, the peptide comprises the sequenceKRWKKFFKKWKGGVIGVTFPF (SEQ ID NO:47), with one, two, three, four, orfive substitutions, insertions, additions, or deletions, with theproviso that the amino acid sequence is not the sequence set forth inSEQ ID NO:2, 3, or 4. In other embodiments, the peptide comprises thesequence KRWKKFFKKWKGGVIGVTFPF (SEQ ID NO:47).

In one embodiment, the peptide comprises the sequenceKRWKKWFKKWKGGVIGVTFPF (SEQ ID NO:48), with one, two, three, four, orfive substitutions, insertions, additions, or deletions, with theproviso that the amino acid sequence is not the sequence set forth inSEQ ID NO:2, 3, or 4. In one embodiment, the peptide comprises thesequence KRWKKWFKKWKGGVIGVTFPF (SEQ ID NO:48).

In another embodiment, the peptide comprises the sequenceKRWKKWWKKWKGGVIGVTFPF (SEQ ID NO:49), with one, two, three, four, orfive substitutions, insertions, additions, or deletions, with theproviso that the amino acid sequence is not the sequence set forth inSEQ ID NO:2, 3, or 4. In another embodiment, the peptide comprises thesequence KRWKKWWKKWKGGVIGVTFPF (SEQ ID NO:49).

In some embodiments, the peptide comprises the sequenceRRWKRFFKRWKGGVIGVTFPF (SEQ ID NO:50), with one, two, three, four, orfive substitutions, insertions, additions, or deletions, with theproviso that the amino acid sequence is not the sequence set forth inSEQ ID NO:2, 3, or 4. In some embodiments, the peptide comprises thesequence RRWKRFFKRWKGGVIGVTFPF (SEQ ID NO:50).

In one embodiment, the peptide of any one of SEQ ID NOs:44-53 isC-terminal amidated.

In other embodiments, the peptides provided herein have a length ofabout 10 amino acids to about 50 amino acids. For example, in someembodiments, a peptide has a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 aminoacids. In other embodiments, a peptide can have a length of, withoutlimitation, about 10 to about 15 amino acids, about 15 to about 20 aminoacids, about 20 to about 25 amino acids, about 25 to about 30 aminoacids, about 30 to about 35 amino acids, about 35 to about 40 aminoacids, about 40 to about 45 amino acids, about 45 to about 50 aminoacids, about 10 to about 20 amino acids, about 20 to about 30 aminoacids, about 30 to about 40 amino acids, or about 40 to about 50 aminoacids.

In some embodiments, a C-terminal amide, or other C-terminal cappingmoiety can be present in peptides described herein. In one embodiment, apeptide as provided herein has a C-terminus that is amidated.

The term “amino acid” as used herein refers to natural amino acids,unnatural amino acids, and amino acid analogs, all in their variousstereoisomers (e.g., D and L stereoisomers or other allostereomers iftheir structures so allow). Natural (or “naturally-occurring”) aminoacids include the 20 “standard” amino acids that are encoded by thecodons of the universal genetic code (alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine), as well as other“non-standard” amino acids that occur naturally but are not encoded bythe codons of the universal genetic code (e.g., hydroxyproline,selenomethionine, and norleucine). Amino acids that are non-standardand/or non-naturally occurring include, without limitation,azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid,beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyricacid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyricacid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminoisobutyricacid, desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid,N-ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylglycine, N-methylisoleucine, N-methylvaline, norvaline,norleucine, ornithine, and pipecolic acid.

An “analog” is a chemical compound that is structurally similar toanother but differs slightly in composition (as in the replacement ofone atom by an atom of a different element or in the presence of aparticular functional group). An “amino acid analog” therefore isstructurally similar to a naturally occurring amino acid molecule as istypically found in native peptides but differs in composition such thateither the C-terminal carboxy group, the N-terminal amino group, or theside-chain functional group has been chemically modified or replacedwith another functional group. Amino acid analogs include natural andunnatural amino acids that are chemically blocked, reversibly orirreversibly, or modified on their N-terminal amino group or theirside-chain groups, and include, for example, methionine sulfoxide,methionine sulfone, S-(carboxymethyl)-cysteine,S-(carboxymethyl)-cysteine sulfoxide and S-(carboxymethyl)-cysteinesulfone. Amino acid analogs may be naturally occurring or can besynthetically prepared. Non-limiting examples of amino acid analogsinclude 5-Hydroxytrpophan (5-HTP), aspartic acid-(beta-methyl ester), ananalog of aspartic acid; N-ethylglycine, an analog of glycine; andalanine carboxamide, an analog of alanine. Other examples of amino acidsand amino acids analogs are listed in Gross and Meienhofer, ThePeptides: Analysis, Synthesis, Biology, Academic Press, Inc., New York(1983).

The stereochemistry of a peptide can be described in terms of thetopochemical arrangement of the side chains of the amino acid residuesabout the peptide backbone, which is defined by the peptide bondsbetween the amino acid residues and the I-carbon atoms of the bondedresidues. In addition, peptide backbones have distinct termini and thusdirection. The majority of naturally occurring amino acids are L-aminoacids (including the 20 standard amino acids as well as a number ofother naturally-occurring, non-standard amino acids), and naturallyoccurring, ribosomally-produced peptides are largely comprised ofL-amino acids. D-amino acids are the enantiomers of L-amino acids.Assembling peptides out of D-amino acids, which are not recognized byproteases, can enable evasion from digestion and remain intact untilreaching membranes (Wade et al., Proc Natl Acad Sci USA87(12):4761-4765, 1990).

The peptides provided herein can be made up of L-amino acids, D-aminoacids, or a combination thereof. For example, in some embodiments, apeptide can have an amino acid composition in which at least about 10%(e.g., at least about 10%, at least about 20%, at least about 25%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%) of the aminoacids are D-amino acids. It is to be noted that some amino acid residueshave more than one stereocenter, and the peptides provided herein can,in some embodiments, include diastereomers of these amino acids thatdiffer from each other only in the configuration of one of theirstereocenters.

In one embodiment, the peptide comprises one or more D-amino acidresidues. In some embodiments, at least about 25 percent,illustratively, about 25 to 100 percent, about 50 to about 55 percent,and about 60 to about 75 percent of the amino acids in the peptide canbe D-amino acids. In one embodiment, at least about 25 percent of theamino acids in the peptide can be D-amino acids. In another embodiment,50 percent of the amino acids in the peptide can be D-amino acids. Inone embodiment, at least about 75 percent of the amino acids in thepeptide can be D-amino acids. In another embodiment, 100 percent of theamino acids in the peptide can be D-amino acids.

In some embodiments, peptidomimetic compounds can be used in place ofthe peptides provided herein. As used herein, the term “peptidomimetic”refers to compounds that are synthetic, non-peptide compounds having athree-dimensional conformation (a “peptide motif”) that is substantiallythe same as the three-dimensional conformation of a selected peptide; apeptidomimetic compound therefore can essentially reproduce elements ofamino acid structural properties and can confer the same or similarfunction as the selected peptide. As compared to a selected peptide, apeptidomimetic compound includes non-naturally occurring modifications,such as an altered backbone and/or non-natural amino acids. In someembodiments, for example, peptidomimetics can include beta-amino acids,peptoids, and/or N-methyl amino acids.

Peptidomimetic compounds can include amide (“peptide”) or non-amide(“non-peptide”) bonds in their backbone structure or can include acombination of peptide and non-peptide bonds in their backbonestructure. Peptidomimetic compounds that are protease resistant or thathave additional characteristics that enhance therapeutic utility, suchas increased cell permeability and prolonged biological half-life, canbe particularly useful. Such compounds typically have a backbone that ispartially or completely non-peptide, but with side groups that areidentical or similar to the side groups of the amino acid residues thatoccur in the peptide upon which the peptidomimetic compound is based.Several types of chemical bonds (e.g., ester, thioester, thioamide,retroamide, sulfonamide, reduced carbonyl, dimethylene andketomethylene) can be useful substitutes for peptide bonds in theconstruction of peptidomimetic compounds. In some embodiments, thecompounds provided herein include hybrids that contain one or morepeptide portions and one or more peptidomimetic portions. Such hybridpeptides can incorporate a combination of natural amino acids andmimetic amino acids (e.g., standard amino acids and peptoids) in thesame molecule.

The peptides provided herein can be obtained by any of a number ofmethods, including those known in the art. In some embodiments, apeptide can be obtained by extraction from a natural source (e.g., fromisolated cells, tissues or bodily fluids), or can be produced byexpression of a recombinant nucleic acid encoding the peptide, or bychemical synthesis (e.g., using solid phase peptide synthesis methods ora peptide synthesizer such as an ABI Peptide Synthesizer; AppliedBiosystems; Foster City, Calif.). For example, standard recombinanttechnology using an expression vector encoding a peptide provided hereincan be used. The resulting peptide then can be purified using, forexample, affinity chromatographic techniques and HPLC. The extent ofpurification can be measured by any appropriate method, including butnot limited to: column chromatography, polyacrylamide gelelectrophoresis, or high-performance liquid chromatography. In someembodiments, a peptide can be designed or engineered to contain a tagsequence that allows the peptide to be purified (e.g., captured onto anaffinity matrix). For example, a tag such as c-myc, hemagglutinin,polyhistidine, or FLAG™ tag (Kodak) can be used to aid peptidepurification. Such tags can be inserted anywhere within the peptide,including at either the carboxyl or amino terminus. Other fusions thatcan be used include enzymes that aid in the detection of the peptide,such as alkaline phosphatase. In some embodiments, a peptide can beamidated at its carboxy terminus.

In some embodiments, a peptide provided herein can be isolated orpurified. A “purified peptide” is a peptide that either has no naturallyoccurring counterpart (e.g., a peptidomimetic), or has been chemicallysynthesized and is thus uncontaminated by other peptides, or has beenrecombinantly produced and has been separated from components of thecell in which it was produced, or that has been separated or purifiedfrom other cellular components by which it is naturally accompanied(e.g., other cellular proteins, polynucleotides, or cellularcomponents). Typically, a peptide is considered “purified” when it is atleast 70%, by dry weight, free from the proteins and other moleculeswith which it naturally associates. A preparation of a purified peptidetherefore can be, for example, at least about 80%, at least about 90%,or at least about 99%, by dry weight, the peptide. Suitable methods forpurifying peptides can include, for example, affinity chromatography,immunoprecipitation, size exclusion chromatography, and ion exchangechromatography. The extent of purification can be measured by anyappropriate method, including but not limited to: column chromatography,polyacrylamide gel electrophoresis, or high-performance liquidchromatography.

In one aspect, the present invention provides a polynucleotide encodinga peptide provided herein, or a nucleic acid molecule (e.g., expressionvector, plasmid, etc.) comprising the polynucleotide encoding thepeptide.

In other aspects, the activities of the peptides provided herein can betested using any of a number of suitable methods, including thosedescribed in the Examples herein.

An activity of a peptide against bacteria, for example, can be tested byculturing the bacteria in a suitable liquid medium until cells reach adesired density (e.g., OD₆₀₀ of 0.8 to 1.1), and then diluting the cellsto a suitable concentration in buffer containing varying concentrationsof one or more selected peptides. Peptide concentrations used in theassays can range from 0 μg/ml to about 100 μg/ml with intermediateconcentrations (e.g., about 0.01 μg/ml, about 0.05 μg/ml, about 0.1μg/ml, about 0.5 μg/ml, about 1 μg/ml, about 2.5 μg/ml, about 5 μg/ml,about 7.5 μg/ml, about 10 μg/ml, about 25 μg/ml, about 50 μg/ml, 75μg/ml, about 0.01 μg/ml to about 0.1 μg/ml, about 0.05 μg/ml to about0.5 μg/ml, about 0.1 to about 1 μg/ml, about 0.5 μg/ml to about 5 μg/ml,about 2.5 μg/ml to about 10 μg/ml, or any other range between about 0.01μg/ml and about 100 μg/ml) that vary for each peptide in order tomaximize the number of data points. Assay cultures can be incubated fora desired length of time (e.g., about two hours), and serial dilutionsof each sample can be prepared and plated. After a suitable length ofincubation, colonies can be counted to determine the effectiveness ofthe peptide(s).

Bacterial survival at each peptide concentration can be calculatedaccording to the ratio of the number of colonies on the platescorresponding to the peptide concentration and the average number ofcolonies observed for assay cultures lacking peptide. The peptideconcentration required to kill about 50% of the viable cells in theassay cultures (EC₅₀) can be determined by plotting percent survival asa function of the log of peptide concentration (log μg/ml) and fittingthe data to Equation (1) using, for example, GraphPad Prism (GraphPadSoftware, Inc., San Diego, Calif.), which describes a sigmoidaldose-response.

S=S _(B)((S _(T) −S _(B))/(1+10^((LogEC50-X)H)))  (1)

In Equation (1), S is percent survival, S_(T) and S_(B) represent theupper and lower survival boundaries, X is the log of the peptideconcentration, and H is the Hill slope of the transition region. Anotherform for Equation (1) is:

Y=Bottom+((Top−Bottom)/(1+10^([(logEC50-X)*Hill Slope)])))  (1)

where Y corresponds to bacterial survival (in percentage) at a givenpeptide concentration (μg/ml), with X being the logarithm of thatconcentration. In the equation, “Top” and “Bottom” refer to the upperand lower boundaries and were constrained to values <100% and >0%,respectively.

The effect of a peptide on biofilm production can be assessed by, forexample, incubating a biofilm-forming bacteria or other microbe withvarying concentrations of one or more peptides for a certain length oftime (e.g., 24 hours at 37° C.). Optical density of the cultures (e.g.,at OD₆₀₀ nm) can be measured to assess microbial growth, and culturesthen can be stained with crystal violet to assess biofilm production.See, e.g., Durham-Colleran et al., Microb Ecol 59(3):457-465, 2010.

An endotoxin neutralizing activity of a peptide can be assessed by, forexample, the ability of the peptide to inhibit E. coli LPS in a rabbitpyrogenicity test or to increase the lethal dose 50 (LD₅₀) of E. coliLPS in mouse (e.g., CD1 mouse).

In other aspects, the peptides of the invention may be associated withone or more moieties. In some embodiments, the association is covalent.In other embodiments, the association is non-covalent. In oneembodiment, the association is via a terminal (e.g., N-terminus,C-terminus, or both) linker (e.g., an amino acid linker such as e.g.,Lys or Cys) or a chemical coupling agent. In another embodiment, the oneor more moieties can be, e.g., a chemical tag, a solid material (e.g.,nano- or micro-particles), a detectable label, a fusion partner (such asa chemical compound or a peptide), or a substrate e.g., a solid orsemi-solid carrier, support or surface, including a bead (e.g. amicrowell plate, nitrocellulose membrane, beads (e.g., latex,polystyrene)). In some embodiments, the association can be facilitatedby a moiety that has a high affinity to a component attached to thesubstrate, e.g., the peptide can be associated with a biotin moiety, andthe component associated with a surface can be avidin.

In other embodiments, the peptides of the invention are associated with(e.g., covalently or non-covalently attached to) a chemical tag or asolid material (e.g., nano- or micro-particles). In one embodiment, thechemical tag-peptide conjugate or particle-peptide conjugate canassociate with membranes of microbial (e.g., bacterial) or tumor cellswhen brought in contact with a sample comprising the cells and form acomplex, wherein the formation of the complex can be utilized todiagnose, image, isolate and/or determine microbial or tumor cells.

In some embodiments, the peptides of the invention may be conjugated toa detectable label (e.g., dye). In some embodiments, the detectablelabel is a fluorescent label (e.g., fluorescent dye, phosphorescentdye).

In one embodiment, the peptides of the invention may be associated witha molecule or reporter group that is masked such that it can beactivated (i.e. unmasked), for example using light or a chemical agent.

In other embodiments, the peptides of the invention may be associatedwith a fusion partner that can be used to e.g., improve purification, toenhance expression of the peptide in a host cell, to aid in detection,to stabilize the peptide, and the like. Examples of suitable compoundsfor fusion partners include, but are not limited to, polyethylene glycol(PEG), Glutathione-S-transferase, and/or histidine tag.

In another aspect, the present invention provides a compositioncomprising a peptide, or a polynucleotide encoding the peptide, providedherein. In some embodiments, the peptides described herein may beformulated with pharmaceutically acceptable carriers or diluents as wellas any other known adjuvants and excipients in accordance withconventional techniques such as those disclosed in e.g., Remington: TheScience and Practice of Pharmacy, 19^(th) Ed. (Easton, Pa.: MackPublishing Company, 1995); Remington's Pharmaceutical Sciences, 18^(th)Ed. (1990, Mack Publishing Co., Easton, Pa. 18042); Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems,Seventh Ed. (Lippincott Williams & Wilkins 1999).

For example, peptides as provided herein can be formulated incompositions by admixture with one or more pharmaceutically acceptable,non-toxic excipients or carriers. Such compositions can be used to treator prevent microbial infection, for example. In some embodiments, acomposition can include one particular peptide, while in otherembodiments a composition can include two or more different peptides(e.g., peptides having different sequences or different amounts of D-and L-amino acids). In some embodiments, the compositions providedherein can contain one or more peptides at a concentration of about0.001 μg/ml to about 100 μg/ml (e.g., about 0.001 μg/ml to about 0.01μg/ml, about 0.005 μg/ml to about 0.05 μg/ml, about 0.01 μg/ml to about1 μg/ml, about 0.01 μg/ml to about 10 μg/ml, about 0.05 μg/ml to about 5μg/ml, about 0.05 μg/ml to about 25 μg/ml, about 0.1 μg/ml to about 10μg/ml, about 0.5 μg/ml to about 50 μg/ml, about 1 μg/ml to about 100m/ml, or about 10 m/ml to about 100 m/ml.

In some embodiments, the composition further comprises an excipient.Excipients (also referred to as pharmaceutically acceptable carriers)can be liquid or solid and can be selected with the planned manner ofadministration in mind so as to provide for the desired bulk,consistency, and other pertinent transport and chemical properties, whencombined with one or more of peptides and any other components of agiven composition. Common excipients include, without limitation,sterile water, saline, polyalkylene glycols such as polyethylene glycol,oils of vegetable origin, hydrogenated naphthalenes, binding agents(e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose), fillers(e.g., lactose and other sugars, gelatin, or calcium sulfate),lubricants (e.g., starch, polyethylene glycol, or sodium acetate),disintegrates (e.g., starch or sodium starch glycolate), and wettingagents (e.g., sodium lauryl sulfate). In some embodiments,biocompatible, biodegradable lactide polymer, lactide/glycolidecopolymer, polyoxethylene-polyoxypropylene copolymers, or combinationsthereof can be used as excipients for controlling the release of apeptide in vivo.

In other embodiments, a composition can include a peptide and one ormore molecular crowding agents such as, by way of example and notlimitation, FICOLL™ (e.g., FICOLL™ 70), polyethylene glycol (PEG), anddextran. FICOLL™ is a neutral, highly branched, high-mass, hydrophilicpolysaccharide that dissolves readily in aqueous solutions. PEG is apolymer of ethylene oxide and is commercially available over a widerange of molecular weights from 300 g/mol to 10,000,000 g/mol. Dextranis a complex, branched polysaccharide made of glucose molecules. Withoutbeing bound by a particular mechanism, such agents may help to mimic thenatural cellular environment, which may enhance the activity of thepeptide. Such agents can be included in the compositions in amounts fromabout 5% to about 50% wt/vol (e.g., about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, orabout 50% wt/vol, or any range there between, including about 5% toabout 10%, about 10% to about 20%, about 20% to about 25%, about 25% toabout 30%, about 30% to about 40%, or about 40% to about 50%).

In some embodiments, pharmaceutical formulations contemplated for use inthe methods, articles, and kits of the invention may include about 0.01to 1.0% (w/v), in certain embodiments about 0.05 to about 1.0%, of thepeptide, about 0.02 to about 0.5% (w/v) of an acetate, phosphate,citrate or glutamate buffer allowing a pH of the final composition offrom about 3.0 to about 7.0; about 1.0 to 10% (w/v) of a carbohydrate orpolyhydric alcohol tonicifier and, optionally, about 0.005 to 1.0% (w/v)of a preservative selected from the group of m-cresol, benzyl alcohol,methyl, ethyl, propyl and butyl parabens and phenol. In anotherembodiment, such a preservative may be included if the formulatedpeptide is to be included in a multiple use product.

In still further embodiments, a pharmaceutical formulation of thepresent peptides may contain a range of concentrations of thepeptide(s), e.g., between about 0.01% to about 98% w/w, or between about1 to about 98% w/w, or preferably between 80% and 90% w/w, or preferablybetween about 0.01% to about 50% w/w, or more preferably between about10% to about 25% w/w in these embodiments. A sufficient amount of waterfor injection may be used to obtain the desired concentration ofsolution.

In some embodiments, compositions can further include one or more otherpeptides, wherein each of the one or more other peptides has one or morebiological activities (e.g., antimicrobial activity). In one embodiment,the one or more other peptides include, but are not limited to, one ormore cathelicidins. Cathelicidins are known to one of ordinary skill inthe art to refer to a large and diverse collection of cationicantimicrobial peptides, for example as described in U.S. PatentPublication No. 2012-0149631 A1, which is herein incorporated byreference in its entirety.

In one embodiment, compositions also can include one or moreconventional antibiotics (e.g., amoxicillin, cephalexin, bacteriocin,neomycin, and/or polymyxin) and/or active ingredients from wounddressings or wound treatment compositions (e.g., NEOSPORIN®, bacitracin,and silver sulfadiazine).

Compositions can be prepared for topical (e.g., transdermal, sublingual,ophthalmic, or intranasal) administration, parenteral administration(e.g., by subcutaneous, intrathecal, intraventricular, intramuscular, orintraperitoneal injection, or by intravenous drip, in the form of liquidsolutions or suspensions in aqueous physiological buffer solutions), fororal administration (e.g., in the form of tablets or capsules), or forintranasal administration (e.g., in the form of powders, nasal drops, oraerosols), depending on whether local or systemic treatment is desiredand on the area to be treated. Administration can be rapid (e.g., byinjection) or can occur over a period of time (e.g., by slow infusion oradministration of slow release formulations). Compositions for otherroutes of administration also can be prepared as desired usingappropriate methods. In addition, compositions can be prepared for invitro use (e.g., for use on environmental surfaces or on medicaldevices).

Formulations for topical administration of peptides include, forexample, sterile and non-sterile aqueous solutions, non-aqueoussolutions in common solvents such as alcohols, or solutions in liquid orsolid oil bases. Such solutions also can contain buffers, diluents andother suitable additives. Pharmaceutical compositions and formulationsfor topical administration can include transdermal patches, ointments,lotions, creams, gels, drops, suppositories, sprays, liquids, andpowders. Nasal sprays also can be useful, and can be administered by,for example, a nebulizer, an inhaler, or another nasal spray device.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be useful.

Compositions and formulations for oral administration include, forexample, powders or granules, suspensions or solutions in water ornon-aqueous media, capsules, sachets, or tablets. Such compositions alsocan incorporate thickeners, flavoring agents, diluents, emulsifiers,dispersing aids, or binders.

Compositions and formulations for parenteral, intrathecal orintraventricular administration can include sterile aqueous solutions,which also can contain buffers, diluents and other suitable additives(e.g., penetration enhancers, carrier compounds and otherpharmaceutically acceptable carriers).

In other embodiments, the composition is a pharmaceutical composition.

In some embodiments, pharmaceutical compositions can include, but arenot limited to, solutions, emulsions, aqueous suspensions, andliposome-containing formulations. These compositions can be generatedfrom a variety of components that include, for example, preformedliquids, self-emulsifying solids and self-emulsifying semisolids.Emulsions are often biphasic systems comprising of two immiscible liquidphases intimately mixed and dispersed with each other; in general,emulsions are either of the water-in-oil (w/o) or oil-in-water (o/w)variety. Emulsion formulations can be useful for oral delivery oftherapeutics due to their ease of formulation and efficacy ofsolubilization, absorption, and bioavailability.

Liposomes are vesicles that have a membrane formed from a lipophilicmaterial and an aqueous interior that can contain the composition to bedelivered. Liposomes can be particularly useful due to their specificityand the duration of action they offer from the standpoint of drugdelivery. Liposome compositions can be formed, for example, fromphosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoylphosphatidyl-choline, dimyristoyl phosphatidylglycerol, or dioleoylphosphatidylethanolamine. Numerous lipophilic agents are commerciallyavailable, including LIPOFECTIN® (Invitrogen/Life Technologies,Carlsbad, Calif.) and EFFECTENE™ (Qiagen, Valencia, Calif.).

The peptides provided herein further encompass pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, provided herein arepharmaceutically acceptable salts of peptides, prodrugs andpharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. The term “prodrug” indicates a therapeutic agent that isprepared in an inactive form and is converted to an active form (i.e.,drug) within the body or cells thereof by the action of endogenousenzymes or other chemicals and/or conditions. The term “pharmaceuticallyacceptable salts” refers to physiologically and pharmaceuticallyacceptable salts of the peptides described herein (i.e., salts thatretain the desired biological activity of the parent peptide withoutimparting undesired toxicological effects). Examples of pharmaceuticallyacceptable salts include, without limitation, salts formed with cations(e.g., sodium, potassium, calcium, or polyamines such as spermine), acidaddition salts formed with inorganic acids (e.g., hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, or nitric acid), andsalts formed with organic acids (e.g., acetic acid, citric acid, oxalicacid, palmitic acid, or fumaric acid).

Compositions additionally can contain other adjunct components such as,for example, lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, colorings,flavorings, and aromatic substances. When added, however, such materialsshould not unduly interfere with the biological activities of thepeptide components within the compositions provided herein. Theformulations can be sterilized if desired.

Dosing of compositions for administration to a subject typically isdependent on the severity and responsiveness of the condition to betreated, with the course of treatment lasting, in some embodiments, fromseveral days to several months, or in other embodiments until a cure isaffected or a diminution of the condition is achieved. Persons ofordinary skill in the art routinely determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages can vary dependingon the relative potency of individual peptides and can generally beestimated based on EC₅₀ found to be effective in in vitro and in vivoanimal models.

In some embodiments, dosage is about 0.01 μg to about 100 g per kg ofbody weight, and may be given once or more daily, biweekly, weekly,monthly, or even less often. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state.

In some embodiments, a preliminary dosage for human infection can beinferred using guidelines put forth by the FDA (Guidance for Industry:Estimating the Maximum Safe Starting Dose in Initial Clinical Trials forTherapeutics in Adult Healthy Volunteers F.a.D. Administration, Editor.2005 (Rockville, Md.), which is herein incorporated by reference in itsentirety).

In one embodiment, dosage is at least about 0.01 mg per kg of bodyweight, illustratively, about 0.01 mg to about 100 mg per kg of bodyweight, about 0.05 mg to about 50 mg per kg of body weight, about 0.1 mgto about 10 mg per kg of body weight, about 0.4 mg to about 5 mg per kgof body weight, and may be given once or more daily, biweekly, weekly,monthly, or even less often.

In some embodiments, dosage is about 0.4 mg to about 5 mg per kg of bodyweight, and may be given once or more daily, biweekly, weekly, monthly,or even less often.

In other embodiments, a dose of at least about 0.01 μg is given,illustratively, about 0.01 μg to about 1 g, about 0.1 μg to about 0.1 g,about 1 μg to about 24 mg, and may be given once or more daily,biweekly, weekly, monthly, or even less often.

In other embodiments, treatments may differ if a subject is resistant orsuspected of being resistant to certain antibiotics. For example, if asubject has an infection that is resistant to antibiotics, the dose maybe increased, or the treatment may include two or more differentpeptides.

In other embodiments, one or more peptides can be admixed, encapsulated,conjugated or otherwise associated with other molecules, molecularstructures, conventional antibiotics, or mixtures of compounds such as,for example, liposomes, polyethylene glycol, receptor targetedmolecules, or oral, topical or other formulations, for assisting inuptake, distribution, absorption, or activity.

In still another aspect, the present invention provides an article ofmanufacture comprising a peptide as provided herein. In one embodiment,the article is a hygiene product (e.g., a personal hygiene productincluding but not limited to mouthwash and body wash). In anotherembodiment, the article is a wound dressing.

In some embodiments, the article is an invasive device, wherein thepeptide is covalently or non-covalently attached onto a surface of thedevice. Covalent and non-covalent methods for attaching peptides tovarious surfaces are known in the art. In one embodiment, the device isa surgical tool. In another embodiment, the device is an implant. Inother embodiments, the device is a catheter, a staple, a suture, animplant, or a tubing.

In still other aspects, the present invention provides a kit comprisinga peptide provided herein or a polynucleotide encoding the peptide. Inone embodiment, the kit further comprises instructions for using thecomponents contained therein.

In another aspect, the present invention provides a method for treatinginfection by a microbial organism in a subject. The method comprisesadministering to the subject a peptide provided herein or apolynucleotide encoding the peptide. In one embodiment, the peptidecomprises the amino acid sequence of Formula (I) (SEQ ID NO:1). Inanother embodiment, the peptide comprises the amino acid sequence ofFormula (I) (SEQ ID NO:1) with one or more substitutions, insertions,additions, or deletions. In some embodiments, the peptide comprises theamino acid sequence of Formula (I) (SEQ ID NO:1) with one or moresubstitutions, insertions, additions, or deletions and with the provisothat the amino acid sequence is not the sequence set forth in SEQ IDNO:2, 3, or 4. In other embodiments, the peptide comprises the aminoacid sequence of Formula (I) (SEQ ID NO:1) with the proviso that theamino acid sequence is not the sequence set forth in SEQ ID NO:2, 3, or4. In one embodiment, the peptide comprises SEQ ID NO:5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50.

In some embodiments, the infection includes but is not limited toinfections of the gastrointestinal tract, respiratory system,circulatory system, lymphatic system, urinary system, muscular system,skeletal system, nervous system, and reproductive system.

In another embodiment, a method for treating an infection by a microbialorganism is provided, where the method includes contacting the microbialorganism with a peptide or composition as provided herein. In otherembodiments, after the contacting step, growth of the microbial organismcan be reduced by at least about 5 percent, illustratively, about 5percent to 100 percent, about 10 percent to about 99.99 percent, about20 percent to about 95 percent, about 30 percent to about 80 percent,about 40 percent to about 70 percent, and about 50 to about 60 percentwhen measured in an assay to measure colony formation. In someembodiments, after the contacting, growth of the microbial organism canbe reduced by at least about 50 percent when measured in an assay tomeasure colony formation directly or indirectly.

In other embodiments, the infection can be a polymicrobial infection.

In some embodiments, for example, a peptide or a composition comprisingthe peptide as described herein can be used to treat a subject having amicrobial (e.g., bacterial or fungal) infection, such as in a wound thatis in or on a subject (e.g., a mammal such as, without limitation, ahuman). Wounds can result from accidental occurrences, or can resultfrom, for example, medical procedures.

In some embodiments, the subject can be a human who is a medical patient(e.g., a diabetes patient, or a patient in a hospital, clinic, orveterinary setting), a member of the armed services or law enforcement,a fire fighter, or a worker in the gas, oil, or chemical industry. Inone embodiment, the subject is an animal suitable to be treated by aveterinarian including, but not limited to pets and livestock/farmanimals.

In other aspects, the present invention provides a method forpreventing, reducing or inhibiting growth of a microbial organism orbiofilm on a surface. The method comprises contacting the surface with acomposition comprising a peptide provided herein. In one embodiment, thepeptide comprises the amino acid sequence of Formula (I) (SEQ ID NO:1).In another embodiment, the peptide comprises the amino acid sequence ofFormula (I) (SEQ ID NO:1) with one or more substitutions, insertions,additions, or deletions. In some embodiments, the peptide comprises theamino acid sequence of Formula (I) (SEQ ID NO:1) with one or moresubstitutions, insertions, additions, or deletions and with the provisothat the amino acid sequence is not the sequence set forth in SEQ IDNO:2, 3, or 4. In other embodiments, the peptide comprises the aminoacid sequence of Formula (I) (SEQ ID NO:1) with the proviso that theamino acid sequence is not the sequence set forth in SEQ ID NO:2, 3, or4. In one embodiment, the peptide comprises SEQ ID NO:5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50.

In one embodiment, the surface is an environmental surface. In anotherembodiment, the surface is on a prosthetic or an implant. In otherembodiments, the surface is in a living organism (e.g., a human or anon-human animal). In some embodiments, the peptides and compositionsdescribed herein are used in surface coatings for medical devices (e.g.,catheters, prosthetics, implants, and other indwelling devices), or indressings to be applied to a wound on or in a patient.

Biofilms are aggregates of microorganisms in which cells adhere to eachother on a surface. Without wishing to be bound by any particulartheory, it is believed that the adherent cells frequently are embeddedin a self-produced matrix of extracellular polymeric substance (EPS)that generally is composed of extracellular DNA, proteins, andpolysaccharides. Biofilms are ubiquitous, and can form on virtually anynon-shedding, living or non-living surface in a non-sterile aqueous (orvery humid) environment. Biofilms can be found, for example, in natural,industrial, hospital, and veterinary settings. Biofilms can be involvedin a wide variety of microbial infections in the body, including commonproblems such as urinary tract infections, catheter infections, earinfections, formation of dental plaque, gingivitis, coating contactlenses, and less common but more serious conditions such asendocarditis, infections in cystic fibrosis, and infections of permanentindwelling devices such as joint prostheses and heart valves. Bacterialbiofilms also can impair cutaneous wound healing and reduce topicalantibacterial efficiency in healing or treating infected skin wounds.

Chronic opportunistic infections in immunocompromised patients and theaging population are a major challenge for medical professionals, astraditional antibiotic therapies usually are not sufficient to eradicatethe infections. One reason for their persistence seems to be thecapability of the bacteria to grow within biofilms that protect themfrom adverse environmental factors. Pseudomonas aeruginosa is an exampleof an opportunistic pathogen and a causative agent of emergingnosocomial infections. Other examples of microbes that can formmedically relevant biofilms include, without limitation, Streptococcusmutans and Streptococcus sanguinis, which are involved in formation ofdental plaque, Legionella bacteria, and Neisseria gonorrhoeae, which canform biofilms on human cervical epithelial cells.

In some embodiments, after the contacting, growth of the biofilm can bereduced by at least about 5 percent, compared to a control, whenmeasured in an assay to measure optical density. In other embodiments,after the contacting, growth of the biofilm is reduced by at least about25 percent, compared to a control when measured in an assay to measureoptical density.

In other aspects, the present invention provides a method for promotingwound healing in a subject. The method comprises administering to thesubject a peptide or a polynucleotide encoding the peptide. In oneembodiment, the peptide comprises the amino acid sequence of Formula (I)(SEQ ID NO:1). In another embodiment, the peptide comprises the aminoacid sequence of Formula (I) (SEQ ID NO:1) with one or moresubstitutions, insertions, additions, or deletions. In some embodiments,the peptide comprises the amino acid sequence of Formula (I) (SEQ IDNO:1) with one or more substitutions, insertions, additions, ordeletions and with the proviso that the amino acid sequence is not thesequence set forth in SEQ ID NO:2, 3, or 4. In other embodiments, thepeptide comprises the amino acid sequence of Formula (I) (SEQ ID NO:1)with the proviso that the amino acid sequence is not the sequence setforth in SEQ ID NO:2, 3, or 4. In one embodiment, the peptide comprisesSEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50.

In some embodiments, the peptides and compositions described herein canbe used in methods for promoting healing of wounds that are not infected(or that show no evidence of infection). For example, in someembodiments, a peptide or composition comprising one or more peptidesdescribed herein can be useful for treating an uninfected wound in asubject (e.g., a vertebrate such as a human), for example such that thewound has increased numbers of keratinocytes, shrinks in size morerapidly, and/or heals more quickly than it would without administrationof the peptide or composition. In some embodiments, treatment of anuninfected wound with a peptide or composition can be consideredeffective if the wound size is reduced by at least about 5% (e.g., atleast about 10%, at least about 20%, at least about 25%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about90%, or at least about 95%) during or after treatment, as compared to acontrol (e.g., a time point before or earlier in the treatment).

In one aspect, the peptides and compositions also can be used in methodsthat include determining whether a subject having a microbial infectionis resistant to one or more conventional antibiotics (e.g.,methicillin), or is suspected of being resistant to one or moreconventional antibiotics. If the subject is determined to be resistantto the one or more conventional antibiotics or is suspected of beingresistant to the one or more conventional antibiotics, the subject canbe treated with a peptide or composition provided herein. In contrast,if the subject is determined not to be resistant to the one or moreconventional antibiotics or is not suspected of being resistant to theone or more conventional antibiotics, the subject can be treated withthe one or more conventional antibiotics. In such methods, the subjectcan be monitored to determine whether the treatment is effective, andthe treatment can be adjusted accordingly. For example, if the subjectis treated with one or more conventional antibiotics but is subsequentlydetermined to be resistant to the conventional antibiotic(s), thesubject can be treated with a peptide or composition as provided herein.In some embodiments, the subject can be treated with one or more AMPsand conventional antibiotics contemporaneously (e.g., in cases of severeinfection insufficient time to try one or the other treatments).

In another aspect, the peptides and compositions provided herein can beused in methods for improving the effectiveness of treatment formicrobial infection. For example, a method can include administering toa subject an amount of a peptide or composition that issub-anti-microbial but is effective to reduce biofilm levels or inhibitbiofilm formation or administering a peptide under conditions that aresub-anti-microbial but are effective to reduce biofilm levels or inhibitbiofilm formation. For example, a peptide may be less effective as ananti-microbial agent under high salt conditions (e.g., about 125 toabout 150 mM salt, including about 130 mM, about 135 mM, about 140 mM,or about 145 mM salt), but can retain effectiveness as an anti-biofilmagent under such conditions. After one or more sub-anti-microbialtreatments, the subject can be treated with an anti-microbial amount ofthe peptide or composition, with the peptide under conditions that areanti-microbial, or with one or more conventional antibiotics. Thesub-anti-microbial and anti-microbial treatments can be separated by anylength of time, ranging from an hour or less to several hours to a dayor more (e.g., about 0.5 hour, about one hour, about two hours, aboutthree hours, about four hours, about six hours, about 12 hours, about 1day, or more than 1 day). Treatments can be repeated as needed ordesired.

The effectiveness of a peptide or composition containing one or morepeptides as described herein can be determined by assessing microbialgrowth or biofilm growth before, during, and/or after treatment. In someembodiments, for example, samples can be obtained from a subject beforetreatment, and at one or more different time points during or aftertreatment with a peptide or composition as provided herein, andmicrobial growth can be measured by counting the number of colonies thatgrow up from the samples after they are plated on a solid medium or amethod which is a quantitative indication of microbial growth Biofilmgrowth can be measured based on optical density (e.g., at 600 nm) and/orstaining with crystal violet, for example. Treatment with a peptide orcomposition can be considered effective if microbial growth or biofilmformation is reduced by at least about 5% (e.g., at least about 10%, atleast about 20%, at least about 25%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 90%, or at leastabout 95%) during or after treatment, as compared to a control (e.g., atime point before or earlier in the treatment).

Lipopolysaccharide (LPS) is a major structural component of theGram-negative bacterial outer membrane and is believed to protectbacteria from antimicrobial compounds. LPS from E. coli and otherGram-negative bacteria is the endotoxin and, for example, may activateinnate immunity through binding TLR4 receptors. Administration ofparenteral products contaminated with pyrogens including LPS may leadto, for example, development of fever, induction of inflammatoryresponse, shock, organ failure and death in humans or animals.

Also, the outer leaflet of outer membranes of Gram-positive bacteriacontain a special lipid called lipoteichoic acid (LTA).

Without wishing to be bound by any particular theory, it is believedthat the overall positive charge on certain antimicrobial peptides mayassist them to form strong electrostatic interactions with thenegatively charged LPS or other anionic lipids and anionic componentse.g., in the membrane of Gram-negative bacteria neutralizing the overallnegative charge. For example, the binding of such peptides with LPS ofGram-negative bacteria can have a major effect on the stability ofbacterial membranes. Several cationic antimicrobial peptides includingLL-37, SMAP-29, and CAP18 can bind LPS. Some antimicrobial peptides canreduce the host immune response to LPS by binding and sequestering it.

In one aspect, the present invention provides a method for treating orpreventing endotoxemia in a subject. The method comprises administeringto the subject an amount of a peptide effective to bind to an endotoxinso as to treat or prevent endotoxemia in the subject. In one embodiment,the peptide comprises the amino acid sequence of Formula (I) (SEQ IDNO:1). In another embodiment, the peptide comprises the amino acidsequence of Formula (I) (SEQ ID NO:1) with one or more substitutions,insertions, additions, or deletions. In some embodiments, the peptidecomprises the amino acid sequence of Formula (I) (SEQ ID NO:1) with oneor more substitutions, insertions, additions, or deletions and with theproviso that the amino acid sequence is not the sequence set forth inSEQ ID NO:2, 3, or 4. In other embodiments, the peptide comprises theamino acid sequence of Formula (I) (SEQ ID NO:1) with the proviso thatthe amino acid sequence is not the sequence set forth in SEQ ID NO:2, 3,or 4. In one embodiment, the peptide comprises SEQ ID NO:5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50.

In some embodiments, the endotoxemia is associated with endotoxinrelated shock including, but not limited to, septic shock,bacteremia-induced shock, and circulatory shock induced by endotoxin.

In other embodiments, the peptide binds to the endotoxin it encountersin the subject, thereby forming a conjugate that has reduced toxicityand pathogenicity relative to unconjugated endotoxin.

In one embodiment, the peptide binds to the endotoxin it encounters inthe subject but does not cause bacterial lysis so as to preventendotoxin-induced lethality.

In other embodiments, the peptide is covalently or non-covalentlyattached onto a surface of an invasive device, wherein the endotoxincontacts the peptide on the surface of the device during or following aninvasive procedure carried out on the subject.

In one embodiment, the device is a surgical tool.

In another embodiment, the device is an implant.

In other embodiments, the device is a catheter, a staple, a suture, animplant, or a tubing.

In some embodiments, the endotoxin is an LPS of a Gram-negativebacterial strain.

In another embodiment, the bacterial strain is of the genus Francisela,Acinetobacter, Pseudomonas, Klebsiella, Escherichia, Haemophilus,Proteus, Enterobacter, Serratia, Burkholderia, Stenotrophomonas,Alcaligenes, Mycobacterium, Legionella, Neisseria, Yersinia, Shigella,Vibrio, or Salmonella.

In other embodiments, the bacterial strain is of the species Franciselatularensis, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiellapneumoniae, Klebsiella oxytoca, Escherichia coli, Haemophilusinfluenzae, Proteus mirabilis, Enterobacter species, Serratiamarcescens, Burkholderia cepacia, Stenotrophomonas maltophilia,Alcaligenes xylosoxidans, Mycobacterium tuberculosis, Neisseriagonorrhoeae, Yersinia pestis, Shigella dysenteriae, Vibrio cholera, orSalmonella typhi.

In one embodiment, the bacterial strain is of the species Franciselatularensis, Francisela novicida, Francisela hispaniensis, Franciselanoatunensis, Francisela philomiragia, Francisela halioticida, Franciselaendociliophora, Francisela guangzhouensis, or Francisela piscicida.

In another embodiment, the bacterial strain is of the species Franciselatularensis.

In some embodiments, the endotoxin is an LTA of a Gram-positivebacterial strain.

In other embodiments, the bacterial strain is of the genusStaphylococcus, Bacillus, Rhodococcus, Actinobacteria, Lactobacillus,Actinomyces, Clostridium, or Streptococcus.

In some embodiments, the bacterial strain is Staphylococcus aureus,Bacillus anthracis, Streptococcus mutans or Streptococcus sanguinis.

In other aspects, a device coated with a peptide is provided. In oneembodiment, the peptide comprises the amino acid sequence of Formula (I)(SEQ ID NO:1). In another embodiment, the peptide comprises the aminoacid sequence of Formula (I) (SEQ ID NO:1) with one or moresubstitutions, insertions, additions, or deletions. In some embodiments,the peptide comprises the amino acid sequence of Formula (I) (SEQ IDNO:1) with one or more substitutions, insertions, additions, ordeletions and with the proviso that the amino acid sequence is not thesequence set forth in SEQ ID NO:2, 3, or 4. In other embodiments, thepeptide comprises the amino acid sequence of Formula (I) (SEQ ID NO:1)with the proviso that the amino acid sequence is not the sequence setforth in SEQ ID NO:2, 3, or 4. In one embodiment, the peptide comprisesSEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50.

In one embodiment, the device is a surgical tool.

In another embodiment, the device is an implant.

In other embodiments, the device is a catheter, a staple, a suture, animplant, or a tubing.

In another aspect, the present invention provides a method fordetermining an LPS or an LTA in a sample. The method comprisescontacting the sample with a peptide under a condition such that the LPSor the LTS binds to the peptide to form a complex; and detecting thecomplex. In one embodiment, the peptide comprises the amino acidsequence of Formula (I) (SEQ ID NO:1). In another embodiment, thepeptide comprises the amino acid sequence of Formula (I) (SEQ ID NO:1)with one or more substitutions, insertions, additions, or deletions. Insome embodiments, the peptide comprises the amino acid sequence ofFormula (I) (SEQ ID NO:1) with one or more substitutions, insertions,additions, or deletions and with the proviso that the amino acidsequence is not the sequence set forth in SEQ ID NO:2, 3, or 4. In otherembodiments, the peptide comprises the amino acid sequence of Formula(I) (SEQ ID NO:1) with the proviso that the amino acid sequence is notthe sequence set forth in SEQ ID NO:2, 3, or 4. In one embodiment, thepeptide comprises SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

In one embodiment, the sample is a biological fluid sample obtained fromthe subject.

In another embodiment, the sample comprises serum, urine, blood, tissueextract or sputum.

In some embodiments, the sample comprising the LPS or the LTA istransferred onto a suitable support under a condition permitting LPS orthe LTA in the sample to attach to the support prior to contacting thesample with the peptide. In another embodiment, the peptide comprises adetectable label.

In some embodiments, the label comprises a fluorescent moiety, aradioactive moiety, or an enzyme.

In other aspects, the present invention provides a method for diagnosingan LPS- or LTA-associated disorder in a subject. The method comprisesforming a complex between an LPS or an LTA and a peptide. In oneembodiment, the peptide comprises the amino acid sequence of Formula (I)(SEQ ID NO:1). In another embodiment, the peptide comprises the aminoacid sequence of Formula (I) (SEQ ID NO:1) with one or moresubstitutions, insertions, additions, or deletions. In some embodiments,the peptide comprises the amino acid sequence of Formula (I) (SEQ IDNO:1) with one or more substitutions, insertions, additions, ordeletions and with the proviso that the amino acid sequence is not thesequence set forth in SEQ ID NO:2, 3, or 4. In other embodiments, thepeptide comprises the amino acid sequence of Formula (I) (SEQ ID NO:1)with the proviso that the amino acid sequence is not the sequence setforth in SEQ ID NO:2, 3, or 4. In one embodiment, the peptide comprisesSEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50.

In some embodiments, the endotoxin is an LPS of a Gram-negativebacterial strain.

In another embodiment, the bacterial strain is of the genus Francisela,Acinetobacter, Pseudomonas, Klebsiella, Escherichia, Haemophilus,Proteus, Enterobacter, Serratia, Burkholderia, Stenotrophomonas,Alcaligenes, Mycobacterium, Legionella, Neisseria, Yersinia, Shigella,Vibrio, or Salmonella.

In other embodiments, the bacterial strain is of the species Franciselatularensis, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiellapneumoniae, Klebsiella oxytoca, Escherichia coli, Haemophilusinfluenzae, Proteus mirabilis, Enterobacter species, Serratiamarcescens, Burkholderia cepacia, Stenotrophomonas maltophilia,Alcaligenes xylosoxidans, Mycobacterium tuberculosis, Neisseriagonorrhoeae, Yersinia pestis, Shigella dysenteriae, Vibrio cholera, orSalmonella typhi.

In one embodiment, the bacterial strain is of the species Franciselatularensis, Francisela novicida, Francisela hispaniensis, Franciselanoatunensis, Francisela philomiragia, Francisela halioticida, Franciselaendociliophora, Francisela guangzhouensis, or Francisela piscicida.

In another embodiment, the bacterial strain is Francisela tularensis.

In some embodiments, the endotoxin is an LTA of a Gram-positivebacterial strain.

In other embodiments, the bacterial strain is of the genusStaphylococcus, Bacillus, Rhodococcus, Actinobacteria, Lactobacillus,Actinomyces, Clostridium, or Streptococcus.

In some embodiments, the bacterial strain is Staphylococcus aureus,Bacillus anthracis, Streptococcus mutans or Streptococcus sanguinis.

In one embodiment, the LPS or the LTA is present in a sample obtainedfrom the subject.

In another embodiment, the method further comprises obtaining a samplefrom the subject and detecting the complex in the sample.

In one embodiment, the sample is a biological fluid sample obtained fromthe subject.

In another embodiment, the sample comprises serum, urine, blood, tissueextract or sputum.

In some embodiments, the sample comprising the LPS or the LTA istransferred onto a suitable support under a condition permitting the LPSor the LTA in the sample to attach to the support prior to contactingthe sample with the peptide.

In another embodiment, the peptide comprises a detectable label.

In some embodiments, the label comprises a fluorescent moiety, aradioactive moiety, or an enzyme.

In other aspects, the present invention provides a method for treating acomposition comprising an LPS or an LTA. The method comprises contactingthe composition with a peptide under a condition such that the LPS orthe LTA binds to the peptide to form a complex; and separating thecomplex from the composition, thereby reducing or eliminating the LPS orthe LTA from the composition. In one embodiment, the peptide comprisesthe amino acid sequence of Formula (I) (SEQ ID NO:1). In anotherembodiment, the peptide comprises the amino acid sequence of Formula (I)(SEQ ID NO:1) with one or more substitutions, insertions, additions, ordeletions. In some embodiments, the peptide comprises the amino acidsequence of Formula (I) (SEQ ID NO:1) with one or more substitutions,insertions, additions, or deletions and with the proviso that the aminoacid sequence is not the sequence set forth in SEQ ID NO:2, 3, or 4. Inother embodiments, the peptide comprises the amino acid sequence ofFormula (I) (SEQ ID NO:1) with the proviso that the amino acid sequenceis not the sequence set forth in SEQ ID NO:2, 3, or 4. In oneembodiment, the peptide comprises SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

In one embodiment, the composition is for parenteral administration.

In another embodiment, the composition is for oral, intravenous,intramuscular, or subcutaneous administration.

In some embodiments, the composition is a cell culture reagent.

In other embodiments, the composition is blood, plasma, serum, or bonemarrow.

In some embodiments, the endotoxin is an LPS of a Gram-negativebacterial strain.

In another embodiment, the bacterial strain is of the genus Francisela,Acinetobacter, Pseudomonas, Klebsiella, Escherichia, Haemophilus,Proteus, Enterobacter, Serratia, Burkholderia, Stenotrophomonas,Alcaligenes, Mycobacterium, Legionella, Neisseria, Yersinia, Shigella,Vibrio, or Salmonella.

In other embodiments, the bacterial strain is Francisela tularensis,Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae,Klebsiella oxytoca, Escherichia coli, Haemophilus influenzae, Proteusmirabilis, Enterobacter species, Serratia marcescens, Burkholderiacepacia, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans,Mycobacterium tuberculosis, Neisseria gonorrhoeae, Yersinia pestis,Shigella dysenteriae, Vibrio cholera, or Salmonella typhi.

In one embodiment, the bacterial strain is Francisela tularensis,Francisela novicida, Francisela hispaniensis, Francisela noatunensis,Francisela philomiragia, Francisela halioticida, Franciselaendociliophora, Francisela guangzhouensis, or Francisela piscicida.

In another embodiment, the bacterial strain is Francisela tularensis.

In some embodiments, the endotoxin is an LTA of a Gram-positivebacterial strain.

In other embodiments, the bacterial strain is of the genusStaphylococcus, Bacillus, Rhodococcus, Actinobacteria, Lactobacillus,Actinomyces, Clostridium, or Streptococcus.

In some embodiments, the bacterial strain is Staphylococcus aureus,Bacillus anthracis, Streptococcus mutans or Streptococcus sanguinis.

The surface of cancer cell membranes can possess net negative-chargecharacteristics, whereas, normal animal cell membrane-surfaces can belargely neutral in charge characteristics. Accordingly, in otheraspects, the present invention provides for anticancer therapeutics thatare selectively toxic against cancerous cells while relatively less- ornon-toxic against normal cells of a subject.

In one aspect, the present invention provides a method for treating orpreventing a cancer in a subject in need thereof. The method comprisesadministering to the subject a therapeutically or prophylacticallyeffective amount of a peptide provided herein, or a compositioncomprising the peptide or a polynucleotide encoding the peptide. In oneembodiment, the peptide comprises the amino acid sequence of Formula (I)(SEQ ID NO:1). In another embodiment, the peptide comprises the aminoacid sequence of Formula (I) (SEQ ID NO:1) with one or moresubstitutions, insertions, additions, or deletions. In some embodiments,the peptide comprises the amino acid sequence of Formula (I) (SEQ IDNO:1) with one or more substitutions, insertions, additions, ordeletions and with the proviso that the amino acid sequence is not thesequence set forth in SEQ ID NO:2, 3, or 4. In other embodiments, thepeptide comprises the amino acid sequence of Formula (I) (SEQ ID NO:1)with the proviso that the amino acid sequence is not the sequence setforth in SEQ ID NO:2, 3, or 4. In one embodiment, the peptide comprisesSEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50.

As used herein, the term “treating” does not necessarily imply that asubject is treated until total recovery and includes, for example, theamelioration or management of one or more symptoms of the cancer.“Preventing” the cancer should not be taken to necessarily imply thatcancer development is completely prevented and includes, withoutlimitation, delay of the cancer development.

In one embodiment, the therapeutically effective amount is an amountsufficient to stop or slow the progression of the cancer. In anotherembodiment, the therapeutically effective amount is an amount sufficientto reduce the number of viable cancer cells in the subject. Methods formonitoring the proliferation of cancer cells and progress of cancer in asubject (e.g., tumor size, cell counts, biochemical markers, secondaryindications, etc.) are known to one of ordinary skill in the art.

In some embodiments, the method for treating is used as a co-therapysuch as, for example, administration in conjunction with radiation,surgery, or other chemotherapeutics. In other embodiments, the methodincludes administration of a therapeutically effective amount of thepeptide in combination with an additional anti-cancer agent. A widevariety of anti-cancer (i.e., anti-neoplastic) agents are known in theart and include, for example alkylating agents, antimetabolites, naturalantineoplastic agents, hormonal antineoplastic agents, angiogenesisinhibitors, differentiating reagents, RNA inhibitors, antibodies orimmunotherapeutic agents, gene therapy agents, small molecule enzymaticinhibitors, biological response modifiers, and anti-metastatic agents.

In still further embodiments, the method for treating can be used anadjuvant therapy such as, for example, administering after surgery orother treatments (e.g., radiation, hormone therapy, or chemotherapy).Accordingly, in such embodiments, the method of adjuvant therapyencompasses administering following a primary or initial treatment, andeither alone or in combination with one or more other adjuvanttreatments, including, for example surgery, radiation therapy, orsystemic therapy (e.g., chemotherapy, immunotherapy, hormone therapy, orbiological response modifiers). In other embodiments, the method relatesto neoadjuvant therapy, which is administered prior to a primarytreatment.

Some non-limiting examples of cancer include carcinoma, melanoma,lymphoma, blastoma, sarcoma, germ cell tumors, and leukemia or lymphoidmalignancies. Non-limiting examples of cancers that fall within thesebroad categories include squamous cell cancer (e.g., epithelial squamouscell cancer), lung cancer including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung and squamous carcinoma ofthe lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including lung cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, cancer of the urinary tract, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain, aswell as head and neck cancer, and associated metastases.

In other embodiments, cancer also encompasses cell proliferativedisorders which are associated with some degree of abnormal cellproliferation and includes tumors, which include neoplasms or neoplasticcell growth and proliferation, whether malignant or benign, and allpre-cancerous and cancerous cells and tissues.

In one embodiment, the cancer is a lung cancer. In some embodiments, thelung cancer is an adenocarcinoma, a squamous cell carcinoma, a largecell carcinoma, a small cell lung cancer, an adenosquamous carcinoma, ora sarcomatoid carcinoma.

In other aspects, a peptide described herein can be used as a vehicle orcarrier for the targeted delivery of other agents (e.g., antimicrobialor anticancer agents). Targeted delivery is described by e.g., U.S. Pat.No. 8,404,636 B2, which is incorporated herein by reference in itsentirety.

In one aspect, the present invention provides a conjugate comprising apeptide conjugated to an agent, wherein the peptide is connected to theagent directly or through a linker segment, the agent being connected tothe peptide or the linker segment through a stable or cleavable bond,wherein the conjugate carries and facilitates the delivery of theconjugated agent to a microbe or a cancer cell. In one embodiment, thepeptide comprises the amino acid sequence of Formula (I) (SEQ ID NO:1).In another embodiment, the peptide comprises the amino acid sequence ofFormula (I) (SEQ ID NO:1) with one or more substitutions, insertions,additions, or deletions. In some embodiments, the peptide comprises theamino acid sequence of Formula (I) (SEQ ID NO:1) with one or moresubstitutions, insertions, additions, or deletions and with the provisothat the amino acid sequence is not the sequence set forth in SEQ IDNO:2, 3, or 4. In other embodiments, the peptide comprises the aminoacid sequence of Formula (I) (SEQ ID NO:1) with the proviso that theamino acid sequence is not the sequence set forth in SEQ ID NO:2, 3, or4. In one embodiment, the peptide comprises SEQ ID NO:5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50.

In some embodiments, the peptide is amidated at the C-terminus.

In other embodiments, the linker segment affixes the agent to thepeptide through acylation of the amino group of the N-terminus of thepeptide.

The agent may be any compound desired to be delivered to a cell. Suchcompounds include, but are not limited to, those which may provide atherapeutic or diagnostic benefit. In some embodiments, the compoundsare nucleic acids, peptide nucleic acids, polypeptides (including forexample, fusion proteins), carbohydrates, peptidomimetics, smallmolecule inhibitors, chemotherapeutic drugs, anti-inflammatory drugs,antibodies, single chain Fv fragments (SCFV), lipids, proteoglycans,glycolipids, lipoprotein, glycomimetics, natural products, or fusionproteins.

In one embodiment, the agent is an antimicrobial agent.

In some embodiments, the antimicrobial agent is levofloxacin,chloramphenicol, or a diazeniumdiolate.

In other embodiments, the agent is an anticancer agent.

In one embodiment, the anticancer agent is an alkylating agent, anantimetabolite, a natural antineoplastic agent, a hormonalantineoplastic agent, an angiogenesis inhibitor, a differentiatingreagent, a RNA inhibitor, an antibody or immunotherapeutic agent, a genetherapy agent, a small molecule enzymatic inhibitor, a biologicalresponse modifier, or an anti-metastatic agent.

In some embodiments, the peptide and the agent to be delivered to a cellare connected to each other to allow the peptide to carry the compoundacross a cell membrane into a cell. Forms of attachment are known in theart and include, without limitation, bonding, fusion or associationbetween the carrier peptide and the at least one compound (for example,but not limited to, covalent bonding, ionic bonding, hydrogen bonding,aromatic stacking interactions, amide bonds, disulfide bonding,chelation). In some embodiment, the carrier peptide and the agent may beconnected in an irreversible or a reversible manner, such that uponentry into a cell the agent is released from the carrier peptide.

In other embodiments, the agent is connected to the carrier peptide atits N-terminus, its C-terminus or at any other location. In oneembodiment, the agent is connected to the peptide at its N-terminus. Inanother embodiment, the agent is connected to the peptide at itsC-terminus. In other embodiments, the agent is connected to the peptidevia a linker molecule. In some embodiment, the linker molecule is apeptide linker.

In other aspects, the invention provides a method for targeting deliveryof the agent to a cell, the method comprising administering to a subjecta therapeutically or prophylactically amount of a conjugate describedherein.

In one embodiment, the cell is a bacterial cell, a fungal cell, or acancer cell.

In another embodiment, the cells are cancer cells; and the inventionprovides a method for targeting delivery of the agent to cancer cells,for example in a mixed population of cancer and non-cancer cells. Insome embodiments, the targeting delivery provides higher selectivityagainst and killing of cancer cells over normal cells.

The peptides and/or conjugates (or nucleic acids or vectors encodingsame) described herein may be delivered to a cell by a number ofdifferent methods known to one of ordinary skill in the art including,but not limited to, in vitro, ex vivo, and/or in vivo methods fordelivery.

In another embodiment, a method for targeting delivery of the inventioncomprises administering the conjugate and/or peptide (or nucleic acidsor vectors encoding same) to a subject.

In one embodiment, an in vitro method may comprise bringing theconjugate and/or peptide (or nucleic acids or vectors encoding same)into contact with one or more cells or a composition comprising one ormore cells or proteins of interest; for example, contacting theconjugate or peptide (or nucleic acids or vectors encoding same) with asample, composition or media in which the one or more cells (or proteinsof interest in certain embodiments) are contained (such as mixing acomposition of the invention with a liquid sample containing one or morecells or proteins).

In another embodiment, an ex vivo method may comprise bringing theconjugate and/or peptide (or nucleic acids or vectors encoding same)into contact with one or more cells or a composition comprising one ormore cells or proteins of interest under a condition that takes placeoutside the subject. For example, in some embodiments, treatment ofimmune cells ex vivo are performed by exposing such cells to theconjugate and/or peptide in an artificial environment (sterileconditions) outside the subject with the minimum alteration of thenatural conditions. In one embodiment, this procedure can involveculturing mononuclear cells that have been isolated from the subjectprior to administration back into the same subject. In some embodiments,the targeting delivery ex vivo provides higher selectivity against andkilling of cancer cells over normal cells that may be administered backinto the subject.

In other aspects, the invention provides a method for delivering theagent to a cell as well as a method for increasing the transferabilityacross cell membrane of the agent to be delivered to the cell, byconnecting the agent of interest to a peptide of the invention. In someembodiments, the peptides described herein may be used as carriers totransport agents across a cell membrane into the cell.

In some embodiments, the method provides forming transient pores in themembrane through which the agent enters the cell.

In one embodiment, the peptide comprises the amino acid sequence ofFormula (I) (SEQ ID NO:1). In another embodiment, the peptide comprisesthe amino acid sequence of Formula (I) (SEQ ID NO:1) with one or moresubstitutions, insertions, additions, or deletions. In some embodiments,the peptide comprises the amino acid sequence of Formula (I) (SEQ IDNO:1) with one or more substitutions, insertions, additions, ordeletions and with the proviso that the amino acid sequence is not thesequence set forth in SEQ ID NO:2, 3, or 4. In other embodiments, thepeptide comprises the amino acid sequence of Formula (I) (SEQ ID NO:1)with the proviso that the amino acid sequence is not the sequence setforth in SEQ ID NO:2, 3, or 4. In one embodiment, the peptide comprisesSEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50.

In some embodiments, the cell is a eukaryotic cell.

In another embodiment, the cell is a bacterial, a fungal or a cancercell.

In some embodiments, the cell is present in a subject and the methodcomprises administering the peptide/agent conjugate/complex to thesubject.

In other embodiments, the cell is ex vivo and the method comprisescontacting the cell with the peptide/agent conjugate/complex.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Materials and Methods

Bacterial Strains. Bacterial strains (Table 1) were obtained fromAmerican Type Culture Collection (ATCC, Manassas, Va.). The bacteriawere first grown in the respective recommended media and then selectedover cation adjusted Mueller Hinton Broth (MHBII, BD 212322) agar(supplemented with antibiotics for antibiotic-resistant strains) andthen grown in MHBII (supplemented with antibiotics forantibiotic-resistant strains). Bacteria were then aliquoted and storedfrozen as 20% glycerol stocks at −80° C.

Peptides. Peptides were procured from several vendors: Anaspec, Inc(Fermont, Calif.), ChinaPeptides, Inc (Shanghai, China) and Virongee(USA). All the peptides were at >95% purity according to themanufacturers' reports.

Reagents and Buffers. Three kinds of phosphate buffers were used such as10 mM Sodium phosphate (Na—PO₄), Phosphate Buffered Saline (PBS) withoutcalcium or magnesium and Dulbecco's Phosphate Buffered Saline (DPBS)with calcium and magnesium. All the phosphate buffers were adjusted topH=7.4 and sterilized by filtration through 0.22 μm PVDF membrane.Resazurin sodium salt, Nitrocefin, ortho-Nitrophenyl-β-galactoside(ONPG), all the inorganic salts along with Hexadecyltrimethylammoniumbromide (HCMAB), Lipopolysaccharides from Escherichia coli 0111:B4,Lipo-teichoic acid (LTA) from Staphylococcus aureus, Polymyxin B (PMB),(Greiner Bio-one) polypropylene 96 well plates were purchased fromSigma.

Instrumentation and Software. Gemini EM (Molecular Devices) fluorescenceplate reader was used to conduct all the fluorescence reading.Absorbances were measured in (BioTek) Eon. Circular Dichroism studieswere conducted using Jasco J-1500 spectropolarimeter (Jasco, Easton,Md.). Data were pre-analyzed with Excel software before plotting andresults were generated using Graphpad Prism 5 (GraphPad Software, Inc.).

Bio Informatics. ATRA1 and WG12 sequences were manually aligned.Secondary structures of the ATRA1 variants and NA-CATH derived sequenceswere predicted by I-TASSER (Iterative Threading ASSEmbly Refinement)using the deposited NMR structures of crotalicidin as template-sequence.The resulting co-ordinate files (in .pdb format) were visualized by theprograms such as Chimera and Pymol (The PyMOL Molecular Graphics System,Version 2.0 Schrödinger, LLC).

Co-ordinates of LPS were obtained from pdb accession code 1QFG and forATRA1, the predicted structure were used.

Endotoxin Binding. The binding of endotoxin (LPS/LTA) was evaluatedusing a fluorogenic conjugateBODIPY TR-Cadaverine (BC). BC bindsendotoxin loses its fluorescence. The peptide competitively displaces BCfrom endotoxin and the fluorescence is reestablished. Competitiveinhibition of endotoxin-BC binding was utilized due to presence ofpeptides already in complexion with endotoxin. 25 μL of 20 μM ofpeptides in 10 mM Na—PO₄ buffer were incubated with 25 μL of LPS (40μg/mL) or LTA (40 μg/mL) for 15 min and then added with 50 of BC (20uM). Peptides bind to LPS or LTA and competitively prevents BC bind tothe endotoxin. BC loses its fluorescence on binding to endotoxin. Thefluorescence intensity of the unbound BC was measured at excitation=580nm and emission=620 nm.

Circular Dichroism (CD). CD was performed by allowing the samples wereallowed to equilibrate for 5 min at 25° C. in a 1 mm path length quartzcuvette (Jasco) prior to collecting the spectra. Scans were performedfrom 200 to 250 nm at 0.2 nm intervals, with a data integration time of4 s and 1 nm bandwidth. Each test was performed once, and mean residueellipticity (MRE), [0], was calculated from the average of four scans.

$\begin{matrix}{{\lbrack\theta\rbrack\left( {{\deg \cdot {cm}^{2} \cdot d}{mol}^{- 1}} \right)} = \frac{{Ellipticity}{\left( {m\deg} \right) \cdot 10^{6}}}{{{{Pathlength}{}({mm})} \cdot \lbrack{Peptide}\rbrack}{({\mu M}) \cdot \left( {n - 1} \right)}}} & {{Equation}1}\end{matrix}$

where, n=number of residues in the peptide.

The peptides were analyzed at a concentration of 30 μM in 10 mM Na—PO₄,50% (v/v) trifluoroethanol (TFE) in 10 mM Na—PO₄, 17.6 mM sodium dodecylsulfate (SDS) in Na—PO₄ buffer. Some of the peptides were also analyzedin presence of LPS micelles in both 10 mM Na—PO₄ and DPBS at apeptide:lipid ratio of 1:4.2.

Tryptophan Fluorescence Studies. The binding interactions of thepeptides with LPS were determined by using the intrinsic fluorescence ofTrp at excitation wavelength of 280 nm and emission in the range of300-400 nm (Trp Blue Shift). 5 μM of peptides, which contained Trp, weretitrated with increasing concentrations of LPS (0-20 μM). A standardsingle-site binding curve fitted to equation (1) was used to calculatethe binding constants (equilibrium dissociation constant, K_(D)).

f=B _(max) *L*(K _(D) +L)⁻¹  Equation 2:

where, f=fractional saturation of the peptide with respect to LPSexpressed in terms of difference in wavelength,Δλ_(max)=λ_(max)−λ(0)_(max), λ_(max)=emission maxima of the peptide onsuccessive addition of LPS in nm, λ(0)_(max)=emission maxima of thepeptide without addition of LPS in nm, L=ligand (LPS) concentration(04), and K_(D)=equilibrium dissociation constant (μM).

The fluorescence of Trp was quenched by addition of acrylamide (0-0.2 M)to peptides (10 μM) in both free and in LPS (25 μM) bound states inDPBS. Stern-Volmer's constant (K_(sv)) was calculated using equation(2), where F₀=fluorescence intensity in the absence of a quencher,F=fluorescence intensity in the presence of a quencher at eachtitration, and [Q]=concentration of quencher in molarity.

F ₀ /F=1+K _(sv)[Q]  Equation 3:

Antimicrobial Studies. The phosphate buffers used for antimicrobialassays were supplemented with non-nitrogenous energy source, 0.1% (w/v)glucose. To conduct antimicrobial assays, bacterial strains were firstsuspended in respective buffers. E. coli (ATCC 25922) and B. cereus(ATCC 11778) were grown in MHB II up to OD_(600 nm)˜1 and stored asfrozen stock in 20% glycerol at −80° C. Occasionally they were thawedand enumerated by serial dilution and spreading on MHB II agar plates.The concentrations of the frozen stocks of E. coli and B. cereus strainswere 1.0-1.1*10⁸ CFU/mL with OD_(600 nm)˜0.8. In case of other bacterialstrains, they were grown on the days of experiment uptoOD_(600 nm)˜1-1.5 and then adjusted to OD_(600 nm)˜0.8. The growth mediaof the drug-resistant bacterial strains were supplemented with few ofthe respective antibiotics against which they are resistant. 200 μL ofthe 0.8 OD_(600 nm) culture were added to 10 mL of the respective bufferto obtain a suspension which was used to incubate with equal volumes ofpeptide solution in the same buffer. The bacterial strain and the mediaconditions used to culture the bacteria for antimicrobial testing areshown in Table 1.

TABLE 1 List of Microbial Strains and growth conditions (X = nosupplementation) Strain Growth Temp. Bacteria (ATCC ID) MediaSupplements (° C.) Escherichia 25922 MHB-II X 35 coli Escherichia 51659MHB-II 50 μg/mL of each 37 coli of Streptomycin and TetracyclineBacillus 11778 MHB-II X 30 cereus Staphylococcus BAA-1718 MHB-II X 37aureus Acinetobacter 9955 MHB-II X 37 baumannii Acinetobacter BAA-1795MHB-II 10 μg/mL of each 37 baumannii of Ampicillin, Ceftazidime,Gentamycin, Norfloxacin, and 1 μg/mL of Levofloxacin Klebsiella 33495MHB-II X 37 pneumoniae Klebsiella BAA-1705 MHB-II 10 μg/mL of each 37pneumoniae of Ampicillin, Ceftazidime, and Levofloxacin Pseudomonas 9027MHB-II X 37 aeruginosa Candida MYA-2876 YM broth X 37 albicans

Antimicrobial assays in different phosphate buffers and serum wereconducted by adapting, with necessary modifications a fasthigh-throughput method based on Virtual Colony Count technique and latermodified by incorporating the use of resazurin. At first serial dilutionof a microbial strain in respective buffer was poured in the wells of 96well polypropylene microplate and was then added with same volume of2×MHB II supplemented with 80 μg/mL resazurin and incubated at 35° C. ina Gemini EM Microplate Reader (Molecular Devices) overnight withintermittent shaking. As the microbe grows it reduces non-fluorescentresazurin to fluorescent resorufin and the fluorescent intensity of eachwell were measured (excitation=570 nm, emission=590 nm) each 5 min for15 hours. The time (t, min) to reach a certain fluorescence (RFU=20000with respect to the starting fluorescence) was correlated with theinitial bacterial concentration (in CFU/mL) in the particular well toobtain a calibration plot of the form:

Log([Bacteria], CFU/mL)=b+m*t  Equation 4:

where b and m are the intercept and the slope of the correlationdependent on the specific bacterial strain and the buffer used.

To obtain antimicrobial efficacy of the peptides, 50 μL of seriallydiluted peptide in a buffer was incubated with 50 μL microbialsuspension in the same buffer for 3 hours in black polypropylene 96 wellplate. After incubation the wells were added with 100 μL of 2×MHB IIsupplemented with 80 μg/mL resazurin and then incubated with continuousfluorescence reading for overnight time period as described earlier. Thesurviving microbial population (CFU/mL) in each well were calculated byfirst finding the time to reach a fluorescence of RFU=20000 and thecorrelation (4) for the specific microbial strain and buffer. % Survivalrepresents the proportion of viable cells in peptide treated cellsrelative to cells treated with the respective buffer alone.

$\begin{matrix}{\left( {\%{Microbial}{survival}} \right)_{\lbrack{peptide}\rbrack} = {\frac{\begin{matrix}{{Surviving}{population}{after}{incubation}} \\{{with}{{the}\lbrack{peptide}\rbrack}}\end{matrix}}{\begin{matrix}{{Surviving}{population}{after}{incubation}} \\{{with}{no}{peptide}}\end{matrix}}*100}} & {{Equation}5}\end{matrix}$

Finally, the antimicrobial effectiveness of each peptide against theparticular microbial strain in the specific buffer was determined byplotting % microbial survival as a function of the log of theconcentration of the peptide and fitting the resulting data to avariable-slope sigmoidal regression model (equation 6). In thisequation, log(EC₅₀) represents the log of the concentration of thepeptide required to kill half of the microbial population, where S_(min)and S_(max) correspond to the minimal and maximal microbial survivalvalues (respectively), and H, Hill slope, is the parameter defining thesteepness of the transition slopes of sigmoidal survival curves.

% Microbial survival=S _(min)(S _(max) −S _(min))/(1+10^(log(EC) ⁵⁰^()−log[peptide]*H))  Equation 6:

Interactions with Bacterial Membranes. Interaction of the peptides withbacterial membranes in different phosphate buffers were evaluated usingE. coli ML35p, constructed in our laboratory. At first, competent cellsof E. coli ML35 were produced by conventional competent cell preparationmethod, then transformed with pBR322 plasmid. Transformed colonies wereselected after growing on Luria-agar containing 50 μg/mL ampicillin. Asingle transformed colony was grown in presence of ampicillin first inLuria broth and then in MHBII and stored as 20% glycerol stock at −80°C. This frozen stock, on the day of the experiment, was thawed and grownin MHBII containing ampicillin up to OD_(600 nm)=0.8-0.9, washed withPBS and then suspended in the respective buffer up to the requiredOD_(600 nm) for use.

NPN Assay. The outer membrane perturbation of bacteria was evaluated byincubating E. coli ML35p with peptides in presence of NPN. 100 mM NPN inacetone was supplemented proportionately to a bacterial suspension ofOD_(600 nm)˜0.5 to achieve a final concentration of 20 of NPN and themixture was equilibrated for 30 mins. Then, 90 μL of the bacterialsuspension containing 20 μM NPN was added with 10 μL of 250 μM peptide(10×) solution in water and scanned for fluorescence intensity(excitation=350 nm, emission=420 nm). The % Perturbation was calculatedby considering PMB to be causing 100% Perturbation and water (withoutpeptide) as 0% Perturbation. Each peptide was tested in triplicate ineach of the three phosphate buffers used.

Membrane Disruptions. The outer and the inner membrane disruptions bythe peptides were evaluated by incubating bacterial suspension withpeptides and then measuring the activity of enzymes leaked out of thecells. 100 μL of 10× concentration of peptide in water was incubatedwith 900 μL bacterial suspension (OD_(600 nm)˜0.1) in respective bufferfor 3 hours at room temperature with gentle rotation. Then the cells anddebris were separated by centrifugation (10000 r.p.m. for 30 mins) andthe supernatant was collected and tested for activity of enzymes thathad leaked out of the bacterial cells. HCMAB was used as a positivecontrol to cause 100% membrane disruption and water as a negativecausing 0% disruption.

The outer membrane disruption was tested by using the activity ofbeta-lactamase present in the periplasmic space of E. coli ML35p.Peptides when disrupt the outer membrane enough to cause beta-lactamaseto come out, the degree of disruption can be measured by the virtue ofthe kinetics of the enzyme and using a chromogenic cephalosporinsubstrate called nitrocefin. β-lactamase mediated hydrolyzed product ofnitrocefin has an absorbance λ_(max)=486 nm. Supernatant collected fromthe incubation of peptide with bacteria was added with nitrocefinsolution in DPBS and incubated at 35° C. and continuous absorbancereadings. The slope of the linear section of the absorbance curve(absorbance vs time), was used as a measure of membrane disruption andthe % Disruption was calculated as:

$\begin{matrix}{{\%{Outer} - {Membrane}{Disruption}} = {\frac{{Slope}{for}{Nitrocefin}{hydrolysis}{for}{the}{test}{peptide}}{{Slope}{for}{Nitrocefin}{hydrolysis}{for}{HCMAB}}*100}} & {{Equation}7}\end{matrix}$

The inner membrane disruption was tested by using the activity ofβ-galactosidase present in the periplasmic space of E. coli ML35p, whichlacks lactose-permease. Peptides when disrupt the inner membrane enoughto cause β-galactosidase to come out, the degree of disruption can bemeasured by the virtue of the kinetics of the enzyme and using asubstrate called ortho-Nitrophenyl-β-galactoside (ONPG). β-galactosidasemediated hydrolyzed product of ONPG has an absorbance λmax=420 nm.Supernatant collected from the incubation of peptide with bacteria wasadded with ONPG solution in DPBS and incubated at 35° C. and continuousabsorbance readings. The slope of the linear section of the absorbancecurve (absorbance vs time), was used as a measure of membrane disruptionand the % Disruption was calculated as:

$\begin{matrix}{{\%{Inner} - {Membrane}{Disruption}} = {\frac{{Slope}{for}{ONPG}{hydrolysis}{for}{the}{test}{peptide}}{{Slope}{for}{ONPG}{hydrolysis}{for}{HCMAB}}*100\%}} & {{Equation}8}\end{matrix}$

Hemolysis. To measure the in-vitro hemolysis activity of the peptides,sheep blood was suspended in DPBS and washed with two times with samebuffer and finally diluted to 20% suspension. Peptides were incubated at125 μM against 10% suspension of sheep blood cells in DPBS for 1 hour at37° C. where DPBS alone served as negative control and 1% TritonX-100served as 100% lysis. After incubation, the cells were pelleted bycentrifugation (1000 r.p.m, 5 min) and the supernatant was transferredto a microplate and read at 540 nm absorbance. Each peptide was testedin triplicate. % Hemolysis was expressed as:

$\begin{matrix}{{\%{Hemolysis}} = {\frac{\left( {Absorbance} \right)_{peptide} - ({Absorbance})_{{no} - {peptide}}}{\left( {Absorbance} \right)_{{TritonX} - 100} - \left( {Absorbance} \right)_{{no} - {peptide}}}*100\%}} & {{Equation}9}\end{matrix}$

Cytotoxicity. Cells were seeded in complete growth medium (MinimalEssential Medium, MEMα, supplemented with 10% Fetal Bovine Serum) into aculture-treated 96-well plate at a density of 25000 cells per well.After 24 hours, culture media was aspirated and replaced with mediacontaining the selected peptide. No-serum experiments were conducted inOpti-MEM I reduced serum media (ThermoFisher, 31985062) and 10% serumexperiments were performed in complete growth medium. In all cases, DBPSwas used as a vehicle only control and buffer to serially dilute thepeptides. After exposure to the peptides or vehicle-alone for thedefined period, Alamar blue dye (resazurin) was added directly to themedia and allowed to incubate for two hours. Following incubation,A570/A600 measurements were made to calculate reduction of resazurin dyeto resorufin. A ratio of reduction in treated versus untreated cells wascalculated in order to determine overall cell viability. The human cellswere always maintained in a 37° C. incubator under 5% CO₂.

Example 2 Peptide Design and Properties

Sequence, Hydrophobic Moment and Wimley-White Transfer Energies. Table 2shows primary sequences of the peptides, their molecular weights, TotalHydrophobic Moment and Free Energy of Transfer to phosphatidylcholinemembrane interfaces. Putative helix-forming sequences are bold-faced.The moments and the free energies were calculated using MPEx bypresetting % α-helix=64%, N-terminal protonated, C-terminal amidated forevery peptide and are plotted in FIG. 1 .

TABLE 2 Sequences and physio-chemical properties of peptides CalculatedWimley- Wimley- White White C-terminal  Total Transfer amidated Hydro-free sequence MW phobic energies Peptide Sequence (g/mol) Momentkcal/mol ATRA1 KRFKKFFKKLK 1496.9 6.49 −2.19 (SEQ ID NO: 2) ATRA1-KRAKKFFKKLK 1420.8 5.23 −0.89 A (SEQ ID NO: 4) ATRA1- KKAKKFFKKLK 1392.85.21 −0.71 AK (SEQ ID NO: 5) ATRA1- KRAKKFFKRLK 1448.9 5.18 −1.07 AR(SEQ ID NO: 6) ATRA1- KRAKRFFKRLK 1476.9 5.04 −1.25 AR2 (SEQ ID NO: 7)ATRA1- RRAKRFFKRLK 1504.9 4.86 −1.43 AR3 (SEQ ID NO: 8) ATRA1-RRFKRFFKRLK 1581.0 6.11 −2.73 R3 (SEQ ID NO: 9) ATRA1- RRFRRFFRRLR1665.0 5.87 −3.17 R6 (SEQ ID NO: 10) ATRA1- KRFKKFFKKFK 1530.9 7.05−2.76 F (SEQ ID NO: 11) ATRA1- KRWKKFFKKWK 1609.0 8.47 −4.20 W2(SEQ ID NO: 12) ATRA1- KRWKKWFKKWK 1648.1 8.97 −4.92 W3 (SEQ ID NO: 13)ATRA1- KRWKKWWKKWK 1687.1 9.40 −5.64 W4 (SEQ ID NO: 14) ATRA1-RRWKRFFKRWK 1693.1 8.09 −4.74 R3W2 (SEQ ID NO: 15)

Example 3 Endotoxin Binding

The outer leaflet of the outer membrane of Gram-negative bacteria islargely composed of LPS, a glycolipid. On the other hand, the thickpeptidoglycan layer on the plasma-membrane of Gram-positive bacteriacontains LTA. Both LPS and LTA can trigger massive immune responses inthe form of septic shock in animals and thus act as endotoxins.

The endotoxin binding properties of the peptides were evaluated by theirability to competitively inhibit binding of BODIPY TR-Cadaverine (BC) toLPS and LTA. BC contains two functional moieties: BODIPY(boron-dipyrromethene) and cadaverine connected via a carboxamide.BODIPY is the fluorophore of BC and fluoresces in the free state insolution but loses fluorescence when bound to LPS/LTA. The competitivedisplacement of BC from LPS/LTA by AMPs and thus regeneration of thefluorescence has frequently been used to estimate the binding strengthof AMPs with LPS/LTA. In the present studies, 5 μM of each peptide wasused to displace BC from LPS/LTA. Substitution of Phe₃ with Ala, as inATRA1-A, resulted in decreased LPS binding relative to ATRA1 (FIG. 2 ).Substitution of Arginine₂ with Lys in ATRA1-A resulted in ATRA1-AK,which displayed the highest LPS binding among all the peptides, whereassubstitution of arginine for the Lys residue at the 9^(th) positionafforded ATRA1-AR, which showed the lowest LPS binding of all of thepeptides tested. Such observation contradicts the initial assumptionthat substitution of Lys with arginine would result in increasedefficiency of LPS binding. However, with sequential substitutions of Lyswith arginine, starting with ATRA1-AR, peptide variants show increasedLPS binding with each additional arginine substitution. In terms of LPSbinding: ATRA1-AR<ATRA1-AR2<ATRA1-AR3. In ATRA1-R3, the lysine residuesat 9th, 5^(th) and 1^(st) positions of ATRA1 were substituted witharginine. In ATRA1-R6 all of the lysine residues were replaced witharginine. ATRA1-R3 demonstrated higher LPS binding than ATRA1, andATRA1-R6 bound LPS more effectively than ATRA1-R3. These resultssupported the rational assumption that substitution of lysine witharginine can increase the LPS binding competence in ATRA1-basedpeptides.

The importance of the hydrophobicity of the non-polar residues weretested by substituting those residues with other non-polar residues withdifferent degrees of hydrophobicity. When the Phe₃ of ATRA1 wassubstituted with less hydrophobic Ala, the LPS binding decreasedsignificantly. But when Leu₁₀ was substituted with more hydrophobic Phe,the LPS binding also decreased relative to ATRA1 and comparable bindingwas not achieved until all three of the Trp-substitutions for non-polarresidues had been incorporated. Such observations contradict the simpleassumption that replacement of non-polar residues with more hydrophobicones will automatically increase the LPS binding efficiency relative tothe parent peptide. However, such substitutions did increase theLPS-binding efficiency when ATRA1-A, ATRA1-F, ATRA1-W2, ATRA1-W3 andATRA1-W4 were compared, with the trend indicating that substitutions ofnon-polar residues by more hydrophobic ones increased the LPS bindingefficiency within this series of peptides. In ATRA1-W4, all thenon-polar residues were replaced with Trp, and this peptide alonedemonstrated higher efficacy to bind LPS than the parent ATRA1.ATRA1-R3W2, with three arginine substitutions at the 1^(at), 5^(th) and9^(th) positions and two tryptophan substitutions at the 3^(rd) and10^(th) positions, was expected to be more effective in LPS binding thaneither of ATRA1-R3 and ATRA1-W2, which ended up being the case. Based onthese observations, it can be generally inferred, that substitutingarginine for lysine residues and tryptophan for other non-polar residueswithin short helical AMPs, such as ATRA1, can result in increased LPSbinding efficiency. However, BC does not bind LPS effectively in highionic-strength conditions, therefore there is no change in fluorescenceassociated with peptide binding. Therefore, BC displacement cannot beused to monitor LPS binding under high ionic-strength conditions. As aresult, the LPS-binding effectiveness of the peptides could not beobtained in high/physiological saline conditions. The lack of ability toassess LPS-binding results in near physiological ionic conditions usingBODIPY imparts limitations to this study. Despite this limitation, thestudy provides clues into how the cationic nature and hydrophobicity ofresidues impact the LPS binding properties of short AMPs such as ATRA1.

LTA from S. aureus was used to evaluate the LTA binding properties ofthe peptides. All the peptides showed low LTA binding characteristic.However, the general trend of LPS binding among the peptides wasapproximately maintained for LTA binding. ATRA1-AK showed the highestLTA binding. ATRA1-A showed significantly lower LTA binding than ATRA1.LTA binding increased with additional arginine substitutions, whereas,hydrophobic substitutions afforded marginal differences in LTA binding.ATRA1-R3W2 proved to be far effective than ATRA1 in binding LTA. All ofthese results indicate that while certain differences exist in theparameters that determine the binding of the two endotoxins, LPS andLTA, combined substitutions by arginine and tryptophan was found toenhance binding of both the endotoxins.

Example 4 Antimicrobial Efficacies

The antimicrobial efficacies of the peptides were first tested againstthe model Gram-negative bacteria, E. coli ATCC 25922, in differentphosphate buffers having varied ionic strengths and compositions. Thephosphate buffers that were used included 10 mM sodium phosphate(calculated ionic strength ˜25 mM), PBS and DPBS. The PBS used sodiumdihydrogen-phosphate and potassium mono-hydrogen phosphate as bufferingsalts, whereas, added chloride salts of sodium and potassium made theionic strength of the buffer approximately 160 mM (calculated). Theconcentrations of sodium and potassium in PBS was comparable to theirrespective concentrations in serum. DPBS is essentially PBS supplementedwith comparatively minute amounts di-valent cations such as calcium(˜0.9 mM) and magnesium (˜0.45 mM) as chloride salts. Divalent cationsare instrumental in imparting stability to the LPS outer membranesurface in Gram-negative bacteria. PBS is comparable to blood serum interms of ionic strength, whereas the concentrations of calcium andmagnesium in DPBS are almost half that found in serum. However, DPBS isoften used to wash and suspend mammalian cells. Therefore, DPBS providesa buffer system which is physiologically relevant in terms of ionicstrength and composition. Peptides, which are expected to beantimicrobial under physiological conditions, such as in blood, shouldat minimum be effective in DPBS.

All the peptides demonstrated antimicrobial properties in low ionicstrength conditions such as 10 mM phosphate buffer. When the ionicstrength was increased, as in PBS, few of the peptides retained efficacy(FIG. 3 ). ATRA1-AK lost activity in PBS demonstrating the importance ofthe arginine residue at the 2^(nd) position in the ATRA-motif.Surprisingly, ATRA1-AR was completely ineffective while ATRA1-A retainedsome antimicrobial activity, which implies that a single lysine toarginine substitution at the 9^(th) position may be detrimental to ATRApeptide effectiveness. With lysine-to-arginine substitutions at the5^(th) and 9^(th) positions, ATRA1-AR2 is more potent than ATRA1-A.Three lysine-to-arginine substitutions at the 1^(st), 5^(th) and 9^(th)positions improve tolerance for high ionic strength conditions, such asPBS, with ATRA1-AR3 exhibiting greater effectiveness than both ATRA1-Aand ATRA1. In PBS, ATRA1-A was less powerful than ATRA1, while ATRA1-Fwas more effective than ATRA1, demonstrating how increasinghydrophobicity by replacing non-polar residues can contribute to salttolerance in the ATRA peptides. The impact of increasing hydrophobicityby replacing non-polar residues with amino acids that are morehydrophobic, becomes most observable when the peptides were tested inDPBS, which contains minute amounts of divalent cations (Ca⁺⁺ and Mg⁺⁺)added to the PBS. Divalent cations are known to provide stability to theLPS outer sheaf of the OM of Gram-negative bacteria and the degree ofhydrophobicity of the non-polar residues plays a critical role inmodulating the antimicrobial properties exhibited by ATRA-peptides inphysiologically relevant DPBS. ATRA1, ATRA1-A/AR2/AR3, which wereeffective in PBS, lose activity in DPBS. Similarly, ATRA1-F, in whichLeu of ATRA1 was replaced with Phe, was quite effective in PBS but lostactivity in DPBS. The importance of replacing of non-polar residues withTrp becomes evident as such substitutions increased ATRA-motif efficacy,with ATRA1-W4, which incorporates four Trp-substitutions, proving mosteffective in DPBS. The activity shown by ATRA1-R3 was significantlyreduced in DPBS relative to its activity in PBS. Notably, ATRA1-R6, inwhich all of the lysine residues in ATRA1 had been replaced witharginine, was highly effective in DPBS. However, combining arginine andtryptophan substitutions yielded ATRA1-R3W2, which was designed aschimera combining substitutions from both ATRA1-R3 and ATRA1-W2, wasmore effective than either of its parent peptides against E. coli (ATCC25922).

ATRA peptide variants were then tested against B. cereus in DPBS, asmodel Gram-positive bacteria. The peptides were found to be moreeffective against B. cereus. ATRA1 and ATRA1-F, which were ineffectiveagainst E. coli, proved to be somewhat effective against B. cereus inDPBS (FIG. 4 ). However, ATRA1-A and its variants were not effectiveagainst B. cereus under physiologically relevant ionic conditions, whilethe EC₅₀ values of the ATRA1 variants against B. cereus were smaller incomparison to their EC₅₀ values against E. coli in DPBS.

Example 5 Bacterial Membrane Interactions

Some of the ATRA1-variants were further tested for their ability todisrupt outer and inner membranes to form pores or disruptionssufficiently large so as to allow enzymes to leak out of their cellularcompartments (cytoplasm or periplasmic space). For this purpose, tworeporter molecules were used with one being the substrate of acytoplasmic enzyme and the other was a substrate for an enzyme residingin the periplasmic space. One of the reporters, nitrocefin was thesubstrate for β-lactamase, which is present in the bacterial periplasm,whereas the other molecule, ONPG, was the substrate for β-galactosidase,which resides in the cytoplasm of E. coli ML35p strain. So, detection ofβ-lactamase activity in supernatant, collected following incubation withpeptide and subsequent removal of cells, demonstrated outer membranedisruption by formation of pores sufficiently large so as to allow theenzyme β-lactamase to escape the periplasm and enter the externalaqueous environment. Similarly, β-galactosidase activity in thesupernatants is evidence of inner membrane disruption and possible poreformation. When the aforesaid supernatants were tested for nitrocefinand ONPG hydrolysis, the corresponding absorbance increased with time(data not shown) indicating β-lactamase and β-galactosidase activity,respectively, and thus disruption of both the inner and outer membranes.The slopes of the increase in absorbance as a function of time providesthe rate of enzyme activity. The enzyme activities in supernatantscollected after incubation of bacteria with the quaternary ammoniumsurfactant, hexadecyltrimethylammonium bromide (HCMAB) was used as aninternal reference for 100% membrane disruption. Enzyme activity forsupernatants collected for cells incubated in the absence of peptideprovided a reference for 0% membrane disruption. Bee venom peptidemelittin was used as a positive control.

The signals reporting for membrane disruption, especially that ofnitrocefin hydrolysis ((3-lactamase activity), reached maximum intensityvery quickly for cells incubated with peptides in low ionic strength 10mM Phosphate buffer, indicating high degrees of outer membranedisruption (over 90%) relative to HCMAB (FIG. 5 ). However, inphysiologically relevant buffer such as DPBS, the degrees of outermembrane disruption induced by the peptides are varied. While melittinand HCMAB reached maximum signal for nitrocefin hydrolysis withinapproximately 15 mins, ATRA1-R6/R3W2/W3/W4 took approximately 30 mins toreach maximum absorbance. The rate of β-lactamase activity for cellstreated with ATRA1-F in DPBS was intermediate, while those treated withATRA1 exhibited the lowest activity and cells treated with ATRA1-R3demonstrated activity that was in between those observed for ATRA1 andATRA1-F. When the β-lactamase activities associated with the differentpeptides were compared (FIG. 5 ), substitutions of lysine residues witharginine in the ATRA1 peptides proved to lead to greater degrees ofouter membrane disruption. Similarly, substitutions of non-polarresidues in the ATRA1 peptides with more hydrophobic residues resultedin higher degrees of outer membrane disruption in buffer containingphysiologically relevant ionic-composition. Higher outer membranedisruption was observed for ATRA1-R3W2, demonstrating enhancement ofactivity was achieved through simultaneous substitutions of lysineresidues with arginine at the 1^(st), 5^(th) and 9^(th) positions ofATRA1 along with replacement of the two terminal non-polar residues withtryptophan.

The trend for inner membrane disruption differs slightly from thatobserved for outer membrane disruption. The degree of inner membranedisruption is highest for ATRA1 in low ionic strength 10 mM phosphatebuffer, and substitution of lysine residues with arginine or non-polarresidues with more hydrophobic amino acids resulted in decreasedactivity relative to the original ATRA1 peptide (FIG. 5 ). However, whenthe low ionic buffer (10 mM phosphate) was replaced with DPBS, thearginine and hydrophobic substitutions resulted in significantlyincreased activities. The degree of inner membrane disruption caused bythe original ATRA1 peptide fell below 10% relative to HCMAB. As seen inthe other performance assays, ATRA1-R3W2 demonstrated the highest degreeof inner membrane disruption.

Example 6 Bio-Informatics and Design of Peptides

The structure of NA-CATH elucidated by NMR in the presence of bacterialmembrane mimetic anionic liposomes revealed an α-helical segmentstretching from Phe₃ to Lys₂₄, while residues 25-34 lacked any definedstable structure. However, those structures of NA-CATH are unviable inRCSB Protein Data Bank and the I-Tasser suite utilized for predictingthe secondary structures of NA-CATH and its derivatives was made to usethe deposited NMR structures of crotalicidin (PDB ID: 2mwt), asalignment-template. Crotalicidin is a rattlesnake venom-gland peptideand possess high degree of primary sequence similarity with NA-CATH,which is a cobra-snake venom-gland peptide. The secondary structurepredicted for NA-CATH suggested a shorter α-helical segment spanningfrom Phe₃ to Phe₂₁ (Table 3).

TABLE 3^(†) The sequence and predicted secondary structuresSequences / Predicted Secondary Peptides Structure 1 2 3 4 5Crotalicidin KRFKKFFKKVKKSVKKRLKKIFKKPMVIGVTIPF Sequence-(SEQ ID NO: 52) Template NA-CATH KRFKKFFKKLKNSVKKRAKKFFKKPKVIGVTFPF +152668 56 15 29 Sequence (SEQ ID NO: 51) PredictedCCHHHHHHHHHHHHHHHHHHHCCCCCSSSSSCCC Secondary Structure of NA-CATHNA-CATH26 KRFKKFFKKLKNSVKKRAKKFFKKPK +16 3315 73 0 27 Sequence(SEQ ID NO: 53) Predicted CCHHHHHHHHHHHHHHHHHHHCCCCC Secondary StructureATRA1 KRFKKFFKKLK +8 1497 64 0 36 Sequence (SEQ ID NO: 2) ATRA1-RRWKRFFKRWK +8 1693 64 0 36 R3W2 (SEQ ID NO: 15) Sequence PredictedCCHHHHHHHCC Secondary Structure ATRA1-KRFKKFFKKLK-------------GGVIGVTFPF +9 1693 38 24 38 HYD (SEQ ID NO: 38)Sequence ATRA1- RRWKRFFKRWK-------------GGVIGVTFPF +9 2473 38 24 38R3W2-HYD (SEQ ID NO: 50) Sequence PredictedCCHHHHHHHHC-------------CCSSSSSCCC Secondary Structure ^(†)Table 3provides the sequences and predicted secondary structures of the listedpeptides along with their (calculated) molecular weights and expectedcharges at neutral pH. All of the peptides, except NA-CATH, wereamidated at their C-terminii. The secondary structures were predicted byI-Tasser suite using the NMR structure of crotalicidin (PDB ID: 2mwt) asthe template. Non-identical residues in NA-CATH and crotalicidins areshown in bold. The peptide sequences are aligned with NA-CATH and thecorresponding predicted secondary structure for each residue is providedbelow each amino acid. C denotes Coil, H denotes Helix and S denotesSheet as secondary structure element. Column 1: Charge; Column 2:Calculated Mol. Wt. (Da); Column 3: % Residues predicted forminga-helix; Column 4: % Residues predicted forming P-sheet; and Column 5: %Residues predicted forming random coil.

This small discrepancy may be an artifact resulting from the templatesequence, crotalicidin, utilized in predicting NA-CATH secondarystructure. Residues 27-34 (SEQ ID NO:17) of NA-CATH (SEQ ID NO:51) donot contain any polar residue and is referred to as the hydrophobicC-terminal tail (−HYD). The peptides NA-CATH26 and ATRA1 were generatedbased on the N-terminal sequences of NA-CATH with NA-CATH26corresponding to the first 26 residues and ATRA1, the first 11 residues.In order to confer tolerance to the ATRA1 moiety against high saltconditions, the lysines of the first cluster (Lys₅, Lys₉), along withLys₁, was substituted with arginine and the two terminal non-polarresidues (Phe₃ and Lue₁₀) were replaced with tryptophan, creating thenew peptide variant, ATRA1-R3W2, which demonstrated higher degrees ofbactericidal properties and membrane activity than parent peptide ATRA1.

To evaluate the potent contributions of the elongated hydrophobic tailof NA-CATH to the peptide's interactions with the membranes, we firstgenerated an ATRA1 derivative in which the hydrophobic tail of NA-CATHwas introduced at the C-terminus of ATRA1 by a short -GG- segmentresulting in ATRA1-HYD (SEQ ID NO:38). This peptide showed higherantimicrobial and membrane disruption activities than ATRA1. Thehydrophobic tail was next introduced into the ATRA1-R3W2 in a similarfashion. Comparisons of activities of NA-CATH and NA-CATH26 demonstratedhow the absence of the C-terminal elongated tail impacted the peptideactivities in the context of the parent peptide. NA-CATH, ATRA1-HYD andATRA1-R3W2-HYD (SEQ ID NO:50) are predicted to form partial α-helicalconformations. Residues Phe₃ to Lys₂₁ in NA-CATH are predicted to behelical, while the C-terminal five residues 27-31 adopt strand form,flanked by residues 22-26, and residues 32-34 forming coils. In thepredicted secondary structures of ATRA1-HYD and ATRA1-R3W2-HYD, residues3-10 are predicted to be helical, residues 11-13 random coil, residues14-19 β-strand and the terminal 3 residues are random coil again.NA-CATH26 is predicted to form a relatively straight α-helixencompassing Phe₃-Phe₂₁, while the terminal residues Lys₁-Arg₂ andPhe₂₂-Lys₂₆ are supposed to be random coil.

The basic residues of the helical segment are predicted to interact withthe anionic phosphate groups of bacterial lipids, such as LPS, while thenon-polar residues on the opposite hydrophobic face are inserted intothe hydrophobic interior and engage in hydrophobic interactions with thelipid acyl chains. The hydrophobic tails were also hypothesized toenhance the lipid binding, bacterial membrane disruption and consequentantimicrobial effects exerted by the peptides, akin to themethyl-octanoate segment of PMB. On the other hand, PMN which lacks themethyl-octanoate segment show very low activity.

Example 7 Endotoxin Binding

LPS and LTA are abundant membrane components of Gram-negative andGram-positive bacterial membranes and are among the first molecules thatpeptides encounter before destabilizing the bacterial membranes. Hencethe peptides were evaluated for their ability to bind endotoxin bytesting their competitive displacement of cationic BODIPY TR-Cadaverine(BC) from LPS/LTA. When bound to LPS/LTA, BODIPY-fluorescence isquenched, but it is restored when the BC is displaced by the peptides.The presence of the -HYD segment in ATRA1-HYD and ATRA1-R3W2-HYDresulted in a ˜2-fold increase in affinity for LPS in both of thepeptides compared to ATRA1 and ATRA1-R3W2 which lacked the hydrophobictail (FIG. 6A). In the low ionic-strength (˜25 mM) buffer used,ATRA1-R3W2 binds LPS marginally greater than ATRA1, whereasATRA1-R3W2-HYD binds LPS with similar affinity as ATRA1-HYD. Deletion ofthe hydrophobic tail from the NA-CATH parent peptide resulted in a ˜30%decrease in affinity for LPS in NA-CATH26. Similar differences inaffinity were also observed for PMB and PMN. This observation suggeststhat deletion of the hydrophobic 6-9 carbon-length fatty acyl group⁹²and a cationic Dab residue from PMB significantly decreased affinity forLPS in PMN.

The LTA binding properties of the peptides were evaluated using LTA fromS. aureus (FIG. 6B). All of the peptides showed low LTA bindingaffinities. The general observed LPS-binding trend was also exhibitedfor LTA-binding. Both ATRA1-HYD and ATRA-R3W2-HYD demonstrated ˜2-foldhigher degrees of LTA binding than their parent peptides, ATRA-1 andATRA1-R3W2 respectively. However, both of the peptides with arginine andtryptophan substitutions showed marginally higher LTA binding than theircorresponding ATRA1 based counterparts. NA-CATH26, which lacks thehydrophobic tail, bound LTA to a significantly lower degree thanNA-CATH, which was similar the case for PMN and PMB. These observationssuggest that the hydrophobic tail in both NA-CATH and the 21-residueATRA-HYD peptides contributes to endotoxin binding.

Example 8 Antimicrobial Efficacies

The antimicrobial efficacies of the peptides were first tested againstmodel Gram-negative bacteria, E. coli ATCC 25922, in different phosphatebuffers with varied ionic strengths and compositions (FIG. 7 ). Thephosphate buffers that were used includes 10 mM sodium phosphate(calculated ionic strength ˜25 mM), PBS (˜160 mM) and DPBS, which is PBScontaining minute amounts of divalent cations, ˜0.9 mM calcium chlorideand ˜0.45 mM magnesium chloride. Divalent cations, such as Ca⁺⁺ andMg⁺⁺, are instrumental in imparting stability to the LPS membrane inGram-negative bacteria. PBS is comparable to blood serum in terms ofionic strength, whereas the concentrations of calcium and magnesium inDPBS are almost half that of serum. However, DPBS is often used to washand suspend mammalian cells. Therefore, DPBS represents a buffer systemwhich is physiologically relevant in terms of ionic strength andcomposition. The peptide effectiveness was then tested in serum againstmultiple pathogenic bacteria, both antibiotic resistant and susceptiblestrains, to evaluate their potential effectiveness in vivo againstbacterial infections. Peptides, which are expected to be antimicrobialin physiological conditions, should be effective in serum. When peptidesperformed differently in serum than they did in DPBS, we tested them inDPBS supplemented with 4% (w/v) bovine serum albumin, to assess whetherthe differences in performance are due to the presence of high amountsof albumin in serum.

All the peptides demonstrated significant efficacies against E. coli(ATCC 25922) under low ionic conditions (FIG. 7 ). However, under thephysiologically relevant ionic conditions, PMN lost activity and theefficacies of NA-CATH26 and ATRA1-R3W2-HYD decreased approximately2-fold and 5-fold respectively. With further introduction of minuteamounts of divalent cations, in DPBS, ATRA1 lost antimicrobial property,whereas the efficacies of ATRA1-R3W2, ATRA1-HYD and NA-CATH26 decreasedby approximately 3, 10 and 14 folds respectively relative to theirrespective effectiveness in 10 mM Na—PO₄. When tested against E. coli(ATCC 25922) under near physiological ex-vivo condition such as serum,ATRA1-R3W2 demonstrated no antimicrobial property, whereas, the ATRA1and NA-CATH derived peptides, containing the peptide hydrophobic tail,and PMB, containing its acyl-hydrocarbon segment, retained significantantimicrobial activities. To investigate what factors may havecontributed towards the failure of ATRA1-R3W2 in serum while it wasactive in physiologically relevant ionic conditions, we tested thepeptides in a new buffer condition: DPBS supplemented with 4% (w/v)bovine serum-albumin (BSA). Serum contains serum-albumin other thaninorganic salts of Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺. While inorganic saltsmaintain the ionic strength, the serum-albumin, which is a majorcomponent (˜4 g/dL in humans) of serum, contributes towards theviscosity of the blood, maintenance of colloidal osmotic pressure,binding of various compounds and contributing significantly in plasmaantioxidant activity and is often used as a blocker to inhibitnon-specific binding in immunochemistry. Serum-albumin possesses astrong net negative-charge and property to bind hydrophobic molecules.It may competitively sequester short amphipathic cationic peptides andthus limit their antimicrobial activities. In the presence of albumin inDPBS, the EC₅₀ values of ATRA1-R3W2, PMB and ATRA1-HYD increasedapproximately by 5, 3 and 2-folds respectively relative to their EC₅₀values in DPBS against E. coli (ATCC 25922). On the contrary,ATRA1-R3W2-HYD and NA-CATH did not demonstrate significant changes inEC₅₀ values in presence of serum-albumin in DPBS. Thus, the presence ofserum-albumin partially explains the loss of activity by short11-residue ATRA1-R3W2 in serum. These results signify the importance ofpresence of hydrophobic segments in peptides for their antimicrobialactivities in near phycological conditions. The new non-natural variant,ATRA1-R3W2-HYD, demonstrated significant antimicrobial effectivenessagainst type strain, E. coli (ATCC 25922), and was further tested fortherapeutic relevance against different microbial pathogens.

ATRA1-R3W2-HYD, NA-CATH and PMB were evaluated for their effectivenessin mammalian serum conditions against pathogenic bacteria of both normaland drug-resistant strains. The main objective of this study was toevaluate the therapeutic potency of the three peptides under nearphysiological ex-vivo conditions. PMB has been adapted in present timesas a last resort drug against antibiotic resistant infections despiteits significant in-vivo cytotoxicity and can serve as a reference forevaluating new peptide drug-candidates. The peptides were tested againstmembers of the ESKAPE pathogens, which have earned considerablenotoriety for causing small molecule antibiotic-resistant infections andconsequent deaths. It is to be noted that the drug-resistant strainswere grown in the presence of some of the respective drugs against whichthey are resistant and tested the peptides on them. The results aresummarized in FIG. 8 . As such, NA-CATH and the new derived variant,ATRA1-R3W2-HYD did not demonstrate antimicrobial effectiveness againstPseudomonas aeruginosa in serum but exhibited good bactericidalproperties under physiologically relevant ionic conditions (DPBS). OnlyPMB was effective against P. aeruginosa in mammalian serum (results notshown). However, ATRA1-R3W2-HYD and NA-CATH were highly effective,though in lower degree than PMB, when tested against all the otherpathogens. Some of the notable pathogens against which ATRA1-R3W2-HYDand NA-CATH were effective in mammalian serum include multi-drugresistant (MDR) Acinetobacter baumannii (ATCC BAA-1795) and Klebsiellapneumoniae (ATCC BAA-1705). In every instance, ATRA1-R3W2-HYD waseffective in a lesser degree than NA-CATH, which may be in part due todecreased net cationic-charge and sequence-length. Nevertheless, theseresults signify the clinical relevance of rationally designed shortervariant ATRA1-R3W2-HYD, which contains the hydrophobic tail of NA-CATH,in combating against MDR bacteria.

Example 9 Bacterial Membrane Disruptions

Peptides were further evaluated for their ability to disrupt bacterialouter and inner membranes and their ability to create pores large enoughto allow enzymes to leak out of the cells. In order to distinguishbetween inner and outer membrane disruption, we assessed for the releaseof two enzymes, β-lactamase and β-galactosidase, which are located indifferent compartments in E. coli ML35p. This strain producesβ-galactosidase in the cytoplasm, but the absence of permease preventslactose transportation across membranes and its fermentation in thecytoplasm. Transformation of E. coli ML35 with pBR322 gives rise to theampicillin-resistant E. coli ML35p strain. The pBR322 plasmid includes agene for β-lactamase and E. coli ML35p produced the ampicillin degradingenzyme β-lactamase, which is localized to the bacterial periplasmicspace. Thus, β-lactamase and β-galactosidase leakage followingincubation with peptides provide indicators of outer-membrane andinner-membrane disruptions, respectively, by formation of openings,pores, in the membranes large enough to allow passage of these enzymes.In this assay, the bacteria, E. coli ML35p, were first incubated inbuffer that contained peptides and the cells were then separated bycentrifugation and the supernatants were tested for β-lactamase andβ-galactosidase activities separately utilizing their respectivesubstrates Nitrocefin, (chromogenic cephalosporin) and ONPG (lactoseanalogue). If the peptides disrupted either membrane, absorbancesresulting from enzyme activities on these substrates would increase withtime and the rate of increase would provide a measure of the rate ofenzyme activity. The enzyme activities in the supernatant collectedfollowing incubation of the bacteria in the presence of a quaternaryammonium surfactant, Hexadecyltrimethylammonium bromide (HCMAB),provided an internal reference for 100% membrane disruptions. The beevenom peptide melittin also provided a positive control.

The signals for Nitrocefin hydrolysis reached their maximum very quickly(within ˜15 min) for peptides incubated in low ionic-strength 10 mMphosphate buffer, and they exhibited similar absorbance slopes over time(data not shown), indicating high degrees of outer membrane disruptions.When the degrees of outer membrane disruption were compared to that ofHCMAB, ATRA1 displayed a higher degree of disruption than ATRA1-R3W2,but a degree of disruption similar to that observed for ATRA1-HYD (FIG.9 ). The other peptides ATRA1-R3W2-HYD, NA-CATH, PMN, PMB and melittindemonstrated similar high degrees of membrane disruption (90-110% w.r.tHCMAB). However, the rates of nitrocefin hydrolysis for peptidesincubated under physiologically relevant ionic strength conditions suchas DPBS varied widely suggesting diverse degrees of outer membranedisruption under these conditions (data not shown). Membrane disruptionby ATRA1 decreased to less than 1% in DPBS (w.r.t. HCAMAB), while thatof ATRA-R3W2 decreased to approximately 4%. Peptide variants thatincluded the hydrophobic tail exhibited higher degrees of outer membranedisruption, with ATRA1-R3W2-HYD demonstrating 160% membrane-disruptionw.r.t. the quaternary-ammonium surfactant HCMAB. PMB demonstrated outermembrane disruption similar to that of ATRA1-R3W2-HYD, while NA-CATH andmelittin afforded slightly lower degrees of membrane-disruption. Underphysiologically relevant ionic strength conditions, absence of thehydrophobic segment resulted in reduced outer membrane disruption, withboth PMN and NA-CATH26 exhibiting significantly lower degrees ofβ-lactamase leakage than observed for PMB and NA-CATH respectively.(However, NA-CATH26 was not tested for outer membrane disruption in lowionic-strength conditions due to the limited amount of peptide that wasavailable).

Example 10 Cytotoxicity

The hemolytic activities of the peptides were tested as an initialmeasure of potential cytotoxicity. The hemolytic properties of thepeptides were tested using sheep erythrocytes and a high peptideconcentration (125 Even at this high concentration, the peptidesdemonstrated very low (<5% w.r.t. 1% TritonX-100) or negligible degreesof hemolysis (FIG. 10 ).

Example 11 Toxicity Against Human Lung Epithelial Cells

The NA-CATH derived peptides and PMB were tested against human lungepithelial cells to evaluate the therapeutic potential of the peptides.Bee venom peptide, melittin, and human cathelicidin, LL37, were used asreference peptides for comparison. Melittin is known to be cytotoxicagainst both normal and cancer cells whereas, LL37 possess tissuespecific activity on cancer/tumor cells and proliferates growth incertain human lung cancer cell lines. The peptides were incubated for aprolonged period of ˜16 hours (overnight) under reduced serumconditions, as presence of serum may hinder killing. Results are shownin FIG. 11 . The degree of resazurin reduction by the cells, afterincubation with the peptides were compared with the degree of resazurinreduction by (control) cells, which were not incubated with any peptide(100% survival) and were regarded as the % survival after incubationwith the respective peptide. Incubation with ATRA1-R3W2-HYD decreasedthe degree of resazurin reduction in the primary cells by 50% w.r.t. thecontrol cells at a concentration ˜50 while NA-CATH did the same at ˜25μM. All the other peptides, including LL37, demonstrated higher degreesof cytotoxicity. Melittin killed the cells at the highest degree. Over10 μM concentration LL37 revealed significant cytotoxicity. Even thoughunexpected, LL37 had demonstrated significant cytotoxicity againstleucocytes and T-cells at the concentrations of 13-25 μM. Significantcytotoxicity of LL37 was observed against cultured lung primaryepithelial cells in the similar concentration range and almost 100%killing at over ˜25 μM concentrations. PMB exhibit significant in vivoand in vitro toxicities and had found initial application in topicalmedications only. However, emergence of antibiotic resistant bacterialinfections has made PMB as one of the drugs of last resort despite itstoxicity. We observed significant toxicity of PMB over ˜6 μMconcentration against the lung primary cells in vitro. The LD₅₀ of PMB,LL37 and Melittin were all below 10 μM. On the contrary, toxicity ofNA-CATH was significant over ˜25 μM concentration and of ATRA-R3W2-HYDat ˜50 μM. All these results suggest NA-CATH and the derived non-naturalvariant, ATRA1-R3W2-HYD, may be clinically more favorable therapeuticswith less toxicity.

Example 12 Toxicity Against Primary and Cancer-Derived Human LungEpithelial Cells

The peptides were tested against both primary and cancer-derived humanlung epithelial cells. H-358 cells were used as model lung epithelialcancer cells. The cells were exposed to the peptides for a brief periodof time (˜6 hours) in presence of 10% fetal bovine serum, which isconducive to proliferation of both the cell types. Under similarconditions of buffer and exposure time, the NA-CATH derived peptidesdemonstrated no cytotoxicity against the primary cells, whereas, LL37exhibited toxicity over ˜10 μM concentration and melittin over ˜1 μM(FIG. 52 ). On the other hand, all the peptides, including LL37demonstrated various degrees of killing of the cancer cells. LL37exhibited toxicity against the H-358 cancer cell line with a LD₅₀˜17 μM.NA-CATH exhibited higher toxicity (LD₅₀˜10.5 μM) against the cancercells in comparison to ATRA1-R3W2-HYD (LD₅₀˜24.5 μM). All these resultssuggest significant selective toxicity of NA-CATH and its derivativeATRA1-R3W2-HYD against human lung cancer epithelial cells even in abrief exposure time under physiologically relevant growth conditions ofthe cells.

Example 13 Perturbation of Cell Membranes

When peptides partition from the bulk aqueous environment to thecellular membranes, they may cause small defects in the membraneorganization, a signature of adsorption of the peptides into themembranes. Such small defects are called membrane perturbations and canbe evaluated by utilizing molecular probes. One such molecular probe isNPN, which is a hydrophobic molecule and is usually repelled to enterthe hydrophobic interiors of the membrane by the polar headgroups ofmembrane-lipids. However, when small defects and gaps are caused duringthe adsorption of the peptides into the membranes, NPN can enter throughthose defects and in the hydrophobic environment of the membraneinterior the molecule fluoresces, which can be measured forquantification of the extent of membrane-perturbation and peptideadsorption in the cellular membrane.

The effects of ionic strengths and compositions on the abilities of thepeptides to perturb membrane integrity (FIG. 13 ) were studied bymonitoring NPN translocation into E. coli membranes (using fluorescence)in presence and absence of peptide under varied buffer conditions. Theextent of membrane perturbation caused by a peptide was normalized withrespect to 25 μM PMB, which was assumed to afford 100% membraneperturbation. At low ionic strength, 10 mM phosphate buffer, all of thepeptides showed substantial perturbation of the outer membrane of E.coli. The extent of membrane perturbation inflicted by a few peptides,such as ATRA1-AR, ATRA1-R6 and ATRA1-F, decreased slightly in high ionicstrength PBS, but in general remained similar to their performance in 10mM phosphate buffer. Dramatic changes in membrane perturbation wereobserved when the peptides were evaluated in DPBS. The presence of thedivalent cations in high ionic conditions blunted membrane perturbationsubstantially for all the peptides tested. The ATRA1-A variants wereparticularly sensitive to the presence of divalent ions (perturbationbelow 40% with respect to PMB). ATRA1-R3/R6/W2/W3/W4/R3W2 exhibitedhigher OM perturbation than ATRA1 in DPBS. ATRA1-R6 showed greaterdegrees of perturbation than ATRA1-R3, and ATRA1-W2/W3/W4 show higherperturbation than ATRA1-F. As was observed for LPS binding, ATRA1-Aproved less effective at perturbing the outer membrane than ATRA-1.These observations demonstrate how substitution of lysine residueresidues with arginine along with replacing nonpolar residues with morehydrophobic amino acids can impact the ability of ATRA peptides toperturb bacterial outer membrane and adsorption of the peptides in themembranes under physiologically relevant ionic conditions.

The effect of the C-terminal hydrophobic tail in the peptides on theperturbation of membranes were evaluated and compared with thehydrophobic acyl segment of Polymyxin (FIG. 14 ). The extent of membraneperturbation by the peptide was normalized with respect to 2504 PMB,which was used as a scale causing 100% membrane perturbation. In lowionic-strength buffer (10 mM Na—PO4), all the peptides demonstratedsignificant membrane perturbations. PMN displayed a little less membraneperturbation while NA-CATH26 and NA-CATH demonstrated higher degree ofmembrane perturbations than PMB. Interestingly, membrane perturbation byATRA-R3W2-HYD was significantly less (75% with respect to PMB) at lowionic conditions but becomes significantly high and comparable to PMB atphysiological ionic-strength conditions. In PBS, Both NA-CATH26 andNA-CATH also perturbed outer membrane greater than PMB, while ATRA1,ATRA1-R3W2, ATRA1-R3W2 perturbed comparable to PMB and the degree ofmembrane perturbation by PMN got decreased to ˜75%. However, strikingdifferences among the degrees of membrane perturbation by the peptideswere observed on supplementing minute amounts of divalent cations tophysiological ionic-strength conditions. In DPBS, degree of membraneperturbation by ATRA1 decreased significantly (less than 50%), whilethat of ATRA-R3W2 was ˜65%, demonstrating that the combination ofarginine and tryptophan-substitutions introduced elevated degree ofmembrane perturbation in 11-residue ATRA-motif. The degrees of membraneperturbation were further increased by addition of the hydrophobic tailto the ATRA1-variants and ATRA1-HYD and ATRA-R3W2-HYD demonstrated ˜75%and ˜100% membrane perturbation w.r.t PMB respectively. However,NA-CATH26 and NA-CATH displayed similar degrees of membraneperturbation, suggesting that deletion of the hydrophobic tail fromNA-CATH did not result in a decrease in membrane-perturbation activity.On the other hand, PMN showed significantly lower membrane perturbationin DPBS (˜35% with respect to PMB). It is worth noting that even thoughATRA1 is a linear peptide and PMN is cyclic, they are both shortpeptides: ATRA1 is 11-residue long while PMN is 9-residue. The resultssuggest that apart from the presence of hydrophobic tail, the peptidelength may also influence the degree of membrane perturbation andadsorption in the membrane.

What is claimed is:
 1. A peptide comprising: (a) the amino acid sequenceset forth in Formula (I) (SEQ ID NO:1):X_(aa1) X_(aa2) X_(aa3) X_(aa4) X_(aa5) X_(aa6) X_(aa7) X_(aa8) X_(aa9) X_(aa10) X_(aa11)

wherein independently of each other: X_(aa1) is Lys or Arg, X_(aa2) isLys or Arg, X_(aa3) is Phe, Ala, or Trp, X_(aa4) is Lys or Arg, X_(aa5)is Lys or Arg, X_(aa6) is Phe or Trp, X_(aa7) is Phe or Trp, X_(aa8) isLys or Arg, X_(aa9) is Lys or Arg, X_(aa10) is Leu, Phe, or Trp, andX_(aa11) is Lys or Arg; or (b) the amino acid sequence set forth inFormula (I) (SEQ ID NO:1) with one substitution or insertion; with theproviso that the amino acid sequence is not KRFKKFFKKLK (SEQ ID NO:2),KRAKKFFKKPK (SEQ ID NO:3), KRAKKFFKKLK (SEQ ID NO:4), or RRFRRFFRRLR(SEQ ID NO: 10).
 2. The peptide of claim 1, wherein: X_(aa3) is Phe,X_(aa6) is Phe, X_(aa7) is Phe, and X_(aa10) is Leu.
 3. The peptide ofclaim 2, wherein X_(aa1) is Arg.
 4. The peptide of claim 2, whereinX_(aa2) is Lys.
 5. The peptide of claim 2, wherein X_(aa4) is Lys. 6.The peptide of claim 2, wherein X_(aa5) is Lys.
 7. The peptide of claim2, wherein X_(aa8) is Lys.
 8. The peptide of claim 2, wherein X_(aa9) isLys.
 9. The peptide of claim 2, wherein X_(aa11) is Lys.
 10. The peptideof claim 2, wherein: X_(aa1) is Arg, X_(aa2) is Lys, X_(aa4) is Lys,X_(aa5) is Lys, X_(aa8) is Lys, X_(aa9) is Lys, and X_(aa11) is Lys. 11.The peptide of claim 10, wherein the peptide comprises (a) the aminoacid sequence set forth in Formula (I) (SEQ ID NO:1).
 12. The peptide ofclaim 1, wherein: X_(aa1) is Lys, X_(aa2) is Arg, X_(aa4) is Lys,X_(aa5) is Lys, X_(aa8) is Lys, X_(aa9) is Lys, and X_(aa11) is Lys. 13.The peptide of claim 12, wherein X_(aa3) is Phe.
 14. The peptide ofclaim 12, wherein X_(aa6) is Phe.
 15. The peptide of claim 12, whereinX_(aa7) is Phe.
 16. The peptide of claim 12, wherein X_(aa10) is Leu.17. The peptide of claim 12, wherein: X_(aa3) is Phe, X_(aa6) is Phe,X_(aa7) is Phe, and X_(aa10) is Leu.
 18. The peptide of claim 17,wherein the peptide comprises (a) the amino acid sequence set forth inFormula (I) (SEQ ID NO:1).
 19. A method for preventing, reducing orinhibiting growth of a bacteria or fungi on a surface or in a fluid, themethod comprising contacting the surface or the fluid with a compositioncomprising the peptide of claim 1, thereby preventing, reducing orinhibiting growth of the bacteria or fungi on the surface or in thefluid.
 20. The method of claim 19, wherein the surface is on aprosthetic or an implant.